Expression of the costimulatory molecule CD28 and cytokines by mast cells

Cytokine expression by murine bone marrow-derived mast cells in response to interleukin-3 (IL-3) treatment and CD28 ligation was investigated. Cytokine uptake and cell-to-cell interactions via cell surface molecule ligations are two factors that may play an important role in how the microenvironment can affect the function and development of resident cells. IL-3, a mast cell growth factor, was observed to affect the cytokine profile of mast cells, and CD28, when crosslinked at the surface of mast cells along with a costimulus, was also observed to affect the cytokine profile of mast cells. Transcript analysis via Reverse Transcriptase-Rapid Polymerase Chain Reaction (RT-RPCR) demonstrated that IL-3 treatment of Stem Cell Factor (SCF)-derived bone marrow mast cells causes an increase in the level of IL-13 transcripts within 30 min and IL-10 transcripts within 1.5 hr. FACscan analysis using intracellular staining indicated that IL-3 derived bone marrow mast cells constitutively express IL-10. FACscan analysis also revealed that SCF-derived bone marrow cells express IL-10 4 days after IL-3 treatment. FACscan analysis also demonstrated that CD28 is expressed constitutively at low levels by IL-3-derived bone marrow mast cells and also expressed at higher levels by SCF-derived bone marrow mast cells at the cell surface after treatment with phorbol myristate acetate (PMA) or either of the bacterial products, lipopolysaccharide (LPS) or the Borrelia burgdorferi-derived outer surface lipoprotein, OspA. Transcript analysis via RT-RPCR determined that crosslinking of CD28 alone at the surface of the IL-3-derived bone marrow mast cells led to an increased level of c-jun transcripts but not c-fos transcripts. Transcript analysis via RT-RPCR also revealed that, with SCF-derived bone marrow mast cells, crosslinking of CD28 simultaneously with treatment of PMA led to increased levels of IL-13 transcripts. These results indicate that mast cells are capable of expressing CD28 and that some portions of the mast cell CD28 signaling pathway are similar to the T cell CD28 signaling pathway. Together, these results demonstrate that mast cells are capable of changing their cytokine profile in response to at least two stimuli within their microenvironment, cytokine uptake (IL-3) and cell surface molecule ligation (CD28).

EXPRESSION OF THE COSTIMULATORY MOLECULE CD28 AND CYTOKINES BY MAST CELLS by Eric Vincent Marietta A dissertation submitted to the faculty of The University of lTtah In partial fulfillment of the requirements of the degree of Doctor of Philosophy In Experimental Pathology Department of Pathology The University of Utah August 1997 THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Eric Vincent Marietta This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. /17 ? , ! i ~woodCasje Lee bpresko I THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Eric Vincent Marietta in its final form and have found that (1) its format, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Approved for the Major Department Approved for the Graduate Council Ann W. Hart Dean of The Graduate School Copyright © Eric Vincent Marietta 1997 All Rights Reserved ABSTRACT Cytokine expression by munne bone marrow-derived mast cells in response to interleukin-3 (IL-3) treatment and CD28 ligation was investigated. Cytokine uptake and cell-to-cell interactions via cell surface molecule ligations are two factors that may play an important role in how the microenvironment can affect the function and development of resident cells. IL-3, a mast cell growth factor, was observed to affect the cytokine profile of mast cells, and CD28, when crosslinked at the surface of mast cells along with a costimulus, was also observed to affect the cytokine profile of mast cells. Transcript analysis via Reverse Transcriptase-Rapid Polymerase Chain Reaction (RT -RPCR) demonstrated that IL-3 treatment of Stem Cell Factor (SCF)-derived bone marrow mast cells causes an increase in the level of IL-13 transcripts within 30 mIn and IL-IO transcripts within 1.5 hr. FACscan analysis uSIng intracellular staining indicated that IL-3 derived bone marrow mast cells consti tutively express IL-IO. FACscan analysis also revealed that SCF-deri ved bone marrow cells express IL-IO 4 days after IL-3 treatment. F ACscan analysis also demonstrated that CD28 is expressed constitutively at low levels by IL-3-derived bone marrow mast cells and also expressed at higher levels by SCF-derived bone marrow mast cells at the cell surface after treatment with phorbol myristate acetate (PMA) or either of the bacterial products, lipopolysaccharide (LPS) or the Borrelia burgdorferi-derived outer surface lipoprotein, OspA. Transcript analysis via RT -RPCR determined that crosslinking of CD28 alone at the surface of the IL-3-derived bone marrow mast cells led to an increased level of c-jun transcripts but not c-fos transcripts. Transcript analysis via RT -RPCR also revealed that, with SCF-derived bone marrow mast cells, crosslinking of CD28 simultaneously with treatment of PMA led to increased levels of IL- 13 transcripts. These results indicate that mast cells are capable of expressing CD28 and that some portions of the mast cell CD28 signaling pathway are similar to the T cell CD28 signaling pathway.' Together, these results demonstrate that mast cells are capable of changing their cytokine profile in response to at least two stimuli within their microenvironment, cytokine uptake (IL-3) and cell surface molecule ligation (CD28). v TABLE OF CONTENTS ABSTRACT ................................................... iv LIST OF FIGlTRES ............................................ viii ACKNOWLEDGMENTS .......................................... x Chapter I. INTRODUCTION .......................................... 1 Mast Cell Associated Diseases ........................ 2 Mast Cell Mediators ................................ 5 Mast Cells and the Innate Immune Response . . . . . . . . . . 6 Cytokine Production ................................ 9 Mas t Cell Lineage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Murine and Human Mast Cell Subclasses ............. 13 Mast Cell Growth Factors ........................... 15 Costimulatory Molecules ........................... 22 CD28 Signaling Pathway ............................ 28 IL-13 ............................................ 30 Introduction to Work in This Thesis ................. 30 References ....................................... 33 II. BONE MARROW-DERIVED MAST CELLS ..................... 48 Abstract .......................................... 4.9 Introduction ...................................... 50 Results ........................................... 53 Discussion ........................................ 68 Experimental Procedures ........................... 69 References ........................................ 73 I I I. MODULATION OF EXPRESSION OF THE ANTI-INFLAMMATORY CYTOKINES INTERLEUKIN-13 AND INTERLEUKIN-10 BY INTERLEUKIN-3 ........................................ 75 Introduction ...................................... 76 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . .77 Results ........................................... 78 Discussion ........................................ 8 1 References ........................................ 82 IV. CD28 EXPRESSION BY MOUSE MAST CELLS IS MODULATED BY LPS AND OSPA LIPOPROTEIN FROM BORRELIA BURGDORFERI. ......................................... 84 Abstract .......................................... 85 Introduction ...................................... 86 Experimental Procedures ........................... 88 Results ........................................... 95 Discussion ....................................... 128 Acknowledgments ................................ 133 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134 V. DISCUSSION ........................................... 140 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4 1 Conclusion ....................................... 149 References ....................................... 152 Vll LIST OF FIGURES Figure 2.1 FACscan analysis of c-kit and IgE receptor expression by 7-week IL-3-derived mouse bone marrow cells ......... 54 2.2 FACscan analysis of c-kit and IgE receptor expression by 2-week IL-3-derived mouse bone marrow cells ......... 56 2.3 FACscan analysis of c-kit and IgE receptor expression by 7-week SCF-derived mouse bone marrow cells .......... 58 2.4 FACscan analysis of c-kit and IgE receptor expression by 3-week SCF-derived mouse bone marrow cells. . . . 60 2.5 Degranu lation by IL-3-deri ved mouse bone marrow cells in response to IgE + antigen ................................ 64 2.6 Degranulation by SCF-deri ved mouse bone marrow cells in response to IgE + antigen ................................ 66 3.1 Bone marrow-derived mast cells are c-kit+ ICD3-. .......... 78 3.2 Constitutive expression of IL-13 transcripts by BM-MMC .............................................. 78 3.3 Activated BM-CTMC express IL-13 transcripts ...... . .79 3.4 IL-3 treatment increases IL-13 transcript levels in BM-CTMC ............................................. 79 3.5 IL-3 treatment increases IL-IO transcript levels in BM-CTMC .............................................. 80 3.6 IL-IO is expressed by BM-derived mast cells .............. 80 3.7 BM-CTMC-like cells were cultured in KL for 3 weeks and then were either cultured with IL-3 for 96 h (B) or without IL-3 (A) .................................... 80 3.8 IL-3 increases IL-13 and IL-I0 transcription in splenocytes and BM cells ................................ 81 3.9 B220+ splenocytes respond differently to IL-3 than B220-............................................ 81 4.1 Analysis of CD28 expression by SCF-derived mast cells via RT-RPCR (A) and FACS(B) ................... 98 4.2 Effects of PMA treatment upon CD28 expression by SCF-deri ved mast cells .............................. 101 4.3 Binding of CD28 by both the 37.51 and PV-l anti-CD28 antisera ..................................... 103 4.4 Effects of LPS treatment upon CD28 expression ............ 106 4.5 Effects of OspA treatment upon CD28 expression. . . . . . . . . . 109 4.6 Analysis via RT -RPCR of CD28 transcript levels after LPS or OspA treatment. ........................... 112 4.7 Comparison of constitutive CD28 and CD14 transcript levels between bone marrow mast cells derived in IL-3 (IL-3MC) and SCF (SCF MC) .............. 115 4.8 Expression of CD28 on unstimulated IL-3-derived mast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .118 4.9 Expression of CD28 on unstimulated PMC ................. 121 4.10 Effects of CD28 crosslinking on c-jun transcript levels in IL-3 derived mast cells ........................ 123 4. 11 Effects of CD28 stimulation on the levels of IL-13, TNF-a, and IL-6 transcripts ........................... 126 ix ACKNOWLEDGMENTS I would like to thank VCH Verlagsgesellschaft mbH for their permission to reprint the article that appears in Chapter III (Marietta, E., Y. Chen, and J. H. Weis. 1996. Modulation of expression of the anti-inflammatory cytokines interleukin-13 and interleukin- 10 by interleukin-3. Eur. 1. Immunol. 26:49). I wish also to express my gratitude to my mentor, John Weis, for his diligence, guidance, and direction in my pursuit of a doctorate. I would like to thank Janis Weis for her advice and suggestions, and I would also like to thank members of both the Weis labs for their help and support throughout the years, especially Tracey Smith and Yiyou Chen. I also would like to thank my parents, Melvin and Sylvia Marietta, and my sister and brother, Sherry and Roger, for their help and support throughout the years. CHAPTER I INTRODUCTION 2 Mast Cell Associated Diseases Mast cells are best known for their association with allergic reactions (hypersensitive immune responses). With these diseases, an individual responds in an atypical fashion to an antigen (allergen). The type of symptoms exhibited in a hypersensitive immune response depend upon the type of allergen and the site of introduction. For example, air-borne allergens are inhaled and enter mucosal areas of the respiratory tracts. For those individuals with an atypical response to an air-borne allergen such as ragweed or pollen, increased levels of histamine, an inflammatory mediator that causes vasodilation, will be released systemically, followed by bronchoconstriction (1). If the allergen is introduced into the skin however, an individual will experience the delayed-type hyper­sensitive reaction (late phase) with the hallmark "wheal and flare" response (2). The wheal and flare response IS first generated with a localized release of histamine and other proinflammatory agents like TNF-a and IL-l followed by an influx of lymphocytes including neutrophils and eosinophils, resulting in further inflammation of the' tissue (3, 4). In a severely atypical individual, the release of histamine and other inflammatory agents is not controlled, resulting in abnormal swelling. Depending on the severity of the reaction, the 3 symptoms may become systemic, resulting In the systemic release of histamine and bronchoconstriction. It was first observed that mast cells were associated with the production of histamine with a study in 1953 by Riley et aI., in which they noted that there was a distinct correlation between the presence of mast cells and the quantity of extractable histamine or heparin from various tissues (5). Most striking was the fact that mastocytomas yielded the most histamine and heparin relati ve to tissue mass. This was a pivotal find, since for the previous 70 years, mast cells had been studied at a histological level. It was from these histological studies, in fact, that these cells were designated "mast" cells. The term "mast" in mast cells was derived from the mast cell's first designation, "mastzellen," made by Paul Ehrlich around 1880 (5, 6). This is a literal description of these cells, in that mastzellen means "fattened" cell in German. This "fattened" nature, or increased size, of the mast cell is due, in part, to the number of large granules within the cell. Between 1880 and 1950 then, these cells were considered an oddity, "fat" cells without.a function. However, once the association with histamine and mast cells was made, the mast cell generated greater interest, with more studies based on the production of histamine by mast cells (5). 4 Later studies focused then on how the mast cell can release such large quantities of histamine in such a short time period. The observation that histamine was released immediately into the circulatory system indicated that the histamine was present in the mast cell in a preformed state and released upon activation of the mast cell. Studies by Bach et al. determined that mast cells could immediately exocytose histamine and other granule contents In response to the crosslinking of the high affinity receptors for IgE or IgO at the surface of the cell, via the binding of specific antigens to the IgE or IgO present in the receptors (7). Because of these results, it was later determined that the mast cell is not actually the dysfunctional element of the atypical individual as previously thought. The dysfunctional element is actually the overproduction of IgE specific against an allergen. Normally, the IgE level in a normal or typical individual is less than 1 J.lg/ml in total circulating serum and is polyclonal. Individuals with severe atopy, though, have an abnormally high level of IgE in their serum (1000 J.lg/ml), due to an abnormal production of IgE that is specific for one antigen (the allergen) (8). This results in mast cells becoming abnormally sensitive to one antigen. An abnormal response to an allergen will occur then, when the allergen is bound by the overabundant type of IgE bound to the surface of the mast cell. Recently, it has also been observed that mast cells, in addition to contributing to allergies, may also be associated with the development of other diseases and pathologies, such as the development of rheumatoid arthritis (9), asthma (10), allergic rhinitis (11), scleroderma (12), and interstitial cystis (13). Of course, some diseases are due to mast cells acting "inappropriately," with increased degranulation occurring. This increased degranulation occurs primarily due to an abnormally high turnover of mast cells and can result In urticaria, syncope, anaphylaxis, and diarrhea (6). Also, vanous types of mastocytomas can lead to similar pathologies. This typically results in sites of inflammation and tissue damage developing at sites of mast cell proliferation (6). Mast Cell Mediators 5 Other inflammatory mediators besides histamine are produced by mast cells and include leukotrienes, prostaglandins, cytokines, and chemokines. These inflammatory agents, once released into the tissue, can cause vasodilation and the recruitment of leukocytes into the local tissue (14). The vasodilation and bronchoconstriction are 6 due to the immediate release of preformed granule components such as histamine, Tumor Necrosis Factor-alpha (TNF-a), leukotrienes, and prostaglandins (1). Leukocyte recruitment and further inflamma-tion are due to the production of cytokines and chemokines (15). Mast cell granule contents can be released Via exocytosis to the surrounding areas as a response to vanous stimuli. One stimulus that induces mast cell degranulation is the above mentioned antigen specific stimulation through the crosslinking of the IgE or IgG receptors at the surface of the mast cell due to cell surface bound IgE or IgG binding to antigen. Other stimuli that induce degranulation include high concentrations of stem cell factor (SCF) (16), the complement proteins C5a and C3a (17), and calcium ionophores, such as A23187 (18). After degranulation, the mast cell has a greatly reduced number of granules and, as such, looks dramatically different than a fully granulated mast cell. The degranulated mast cell is called a "phantom" mast cell, so designated for its newly acquired transparency (19). Mast Cells and the Innate Immune Response The innate immune response, as opposed to the humoral, or antigen specific response, is characterized by the initiation of an 7 Immune response In the absence of T cell help to foreign substances, such as bacteria and bacterial components. One way to initiate an innate immune response is through the escalation of the complement cascade. The complement cascade can be initiated through the classical pathway or the alternative pathway. The alternative pathway can be initiated by the cleavage of C3 into C3a and C3b (20, 21). This cleavage can take place on activating surfaces such as bacteria and viruses. Cleavage of C5 into C5a and C5b will occur later in the cascade. C5a promotes chemotaxis of neutrophils and monocytes/macrophages (22). With leukocyte recruitment, an initial influx of neutrophils occurs within 8 hours followed by an influx of macrophages into the site 8 hours later (23). The above mentioned capability of mast cells to degranulate in response to the complement proteins C5a and C3a indicates an ability of the mast cell to participate in innate immune responses. Indeed, injections of C5a or C3a into humans results in wheal and flare responses with the typical neutrophil recruitment (24, 25). The development of the wheal and flare response is due not only to the ability of C5a and C3a to cause mast cells to degranulate but also their ability to recruit mast cells in a chemotactic fashion (17, 26). Specific receptors for these proteins are expressed by human mast cells as observed by Nilsson et al. (26). 8 Responding to C5a and C3a is not the only example of mast cells being able to participate in the innate immune response. Other examples include the capability of mast cells to bind to enterobacteria. In one study by Malaviya et al. (27), it was observed, that mast cells can adhere to enterobacteria that express the FimH fimbrial protein, phagocytose the enterobacteria and process and present bacterial antigens through MHC Class I. Other studies have observed that mast cells can also respond to the bacterial product lipopolysaccharide (LPS). LPS is produced by gram negative bacteria and is present in the outer membrane of their cell wall (28). Rat peritoneal mast cells treated with LPS will secrete IL- 6 and will release very little histamine (29). Another study observed that rat peritoneal mast cells treated with cholera toxin will increase their IL-6 synthesis and decrease their TNF-a production, without any measurable release of histamine. These data indicate that the mast cells are not degranulating in response to LPS or cholera toxin. However, type 1 fimbriated Escherichia coli can indu'ce mouse mast cell degranulation, resulting in the release of histamine" (31). Thus, mast cells are capable of responding to bacterial products, with some products inducing degranulation, and others inducing different cytokine expression patterns. Another response to LPS by mast cells was observed to be an increased expression of MHC Class II molecules. In that study, bone marrow-derived mast cells, when treated with LPS, upregulated 9 their expression of MHC Class II and were then capable of presenting antigen to MHC Class II restricted T cells. Of interest, was that this upregulation of MHC Class II molecules was observed to be inhibited by treatment with IL-3 or IFN-y. These data suggest that mast cells may be capable of acting as antigen presenting cells in bacterial infections. Cytokine Production The types of cytokines that mast cells express is diverse. These include both inflammatory and anti-inflammatory cytokines, growth factors, and chemokines. For example, the current list includes, but is not limited to, Lymphotactin (Ltn), TNF-a, Transforming Growth Factor-beta (TGF-P), Granulocyte/Monocyte-Colony Stimulating Factor (GM-CSF), Interferon-gamma (IFN-y), Macrophage Inflammatory Protein-l alpha (MIP-l a), Macrophage Inflammatory Protein-l beta (MIP-IP), IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL- 10, IL-12, and IL-13 (33-40). Those cytokines that contribute to a cellular type of immune response are TNF-a, IFN-y, IL-2, and IL-12. 10 This classification is based on the Th 1/Th2 cytokine profile (for review see 41). Those cytokines that contribute to a humoral type of immune response, again based on the Th1/Th2 cytokine profile, are IL-4, IL-10, and IL-13. GM-CSF, IL-5, and IL-3 have been characterized as hemopoietic growth factors (for review see 42). The chemokines produced by mast cells include MIP-1a, MIP-I p, and IL-8 for the human. Cells that respond to these chemokines are macrophages (MIP-I a, MIP-1 P) and neutrophils (IL-8) (for review see 43). IL-5 also serves as a potent chemoattractant for eosino­phils (44). Clearly, this is a wide diversity of cytokines. Either all the cytokines are expressed simultaneously after degranulation, or alternatively, some levels of regulation might exist within the mast cell to control both the timing and the type of cytokines that are released by the mast cell. This latter model, with regulated timing and qualitative release, is supported by observations that the expression or release of cytokines is temporally different for many cytokines. For example, immediately upon degranulation VIa FCER I crosslinking, preformed cytokines, such as TNF-a, are released (45). An hour or two after the degranulation event, other cytokines are transcribed, such as GM-CSF and IL-6 (34). At still later time points, ) 11 4-8 hours, other cytokines are transcribed or their transcript levels increased, such as TNF-a (34). It appears then, that the expression of cytokines and chemokines by mast cells is under some regulation. For the mast cell to produce such a wide diversity of reagents at different time points would also suggest that the mast cell is targeting a large number of different cell types at different times. The receptors for these cytokines are found on T cells, B cells, macrophages, neutrophils, and eosinophils, among other cells. Receptors for IL-2 are found on T cells. Receptors for IL-4, IL-13, and IL-IO are found on B cells and monocytes (for reviews see 47- 48). IL-5 receptors are found on eosinophils, and IL-8 receptors are found on human neutrophils (49). This would suggest that either a very chaotic and unregulated explosive release of cytokines occurs with mast cell degranulation or that a highly regulated temporal series of events occurs, which may be dependent upon signals derived from the local microenvironment in which the mast cell IS localized. Mast Cell Lineage The wide diversity of cytokine expressIon by mast cells suggests a similarity with many cell types, including monocytes, 12 T cells, and B cells. Are mast cells, then, of a monocytic or lymphoid lineage? Because mast cells contain granules, mast cells have been considered to be a typical granulocyte. Therefore, many studies were devoted towards demonstrating that mast cells are of a myeloid lineage, since granulocytes are of the myeloid lineage. Some studies have demonstrated that human monocytes cultured in the presence of fibroblast supernatant gave rise to a cell type that is a gran­ulocyte, expresses IgE receptors, and contains histamine, all mast cell characteristics (50, 51). However, more recent studies have demonstrated that the basophil, which IS more similar to a monocyte than a mast cell, also IS a granulocyte, contains histamine, expresses FCERI, and can release histamine VIa FCERI crosslinking (52). More recent data cataloging the expression of a wide variety of cell surface molecules of the CD classification (CD 11 b, CD 18 among many others) on human mast cells, basophils, and monocytes indicates that human monocytes, basophils, and mast cells are actually three distinct cell types (53). One later study even determined that by isolating mature peripheral monocytes using a monocyte marker, CD14, that mast cells do not develop from these cell types (54). This reopens the question of where in the hematopoietic lineage, myeloid or lymphoid, the mast cell falls. Similarities do exist between mast cells and lymphoid cells. Some similarities between mast cells and T cells include the diverse cytokine/interleukin production and the production of common integrins, such as aM290. aM290 is a molecule expressed by both 13 intraepithelial T cells localized in the mucosa and mucosal-like mast cells (55). There are even studies from the 60s and early 70s where mast cells were cultured from the thymus, peripheral and mesenteric lymph nodes, and thoracic duct lymph by incubating these cells on top of fibroblast monolayers (56-58). Thus, although the mast cell has been considered to be of a myeloid lineage, recent data and older data suggest that the mast cell is a unique cell type, possibly with its own hematopoietic lineage and possibly derived from the precursor to both the myeloid and the lymphoid lineages (54). Murine and Human Mast Cell Subclasses Murine mast cells are categorized into two distinct groups, connective tissue-like mast cells and mucosal-like mast cells. The mast cells that are found in the mucosa are designated as mucosal­like mast cells and those mast cells that are found in connective 14 tissue, such as the skin, peritoneum, and blood and lymphatic vessels are designated as connective tissue-like mast cells (3). The classification of human mast cells is different from the classification of murine mast cells. With human mast cells, the difference between mucosal-like mast cells and connective tissue-like mast cells is less distinct and is based upon the production of two proteases, tryptase and chymase. In the human, mast cells localized in the skin predominantly express chymase with some tryptase (MCTe), whereas mast cells localized in the mucosa predominantly express tryptase and no chymase (MCT) (for review see 59). More distinct differences exist between the munne mucosal­like mast cell and the munne connective tissue-like mast celL One of these differences, among others, is proteoglycan production. Chondroitan sulfate proteoglycan IS produced by mucosal-like mast cells but not connective tissue-like mast cells. Heparin sulfate however, is produced by murine connective tissue-like mast cells but not mucosal-like mast cells (59). Another difference between the two types of cells include histological staining. Mucosal-like mast cells are alcian blue positive, safranin red negative, whereas connective tissue-like mast cells are safranin red positive, alcian blue negative (59). Another difference is the level of histamine within each cell, where the connective tissue-like mast cell contains high 15 levels of histamine and the mucosal-like mast cell contains low levels of histamine (59). Other important differences between the two types of munne mast cells exist in the production of cytokines. One study by Smith et al. (39) observed constitutive expression of IL-4 by IL-3-deri ved bone marrow mast cells, which resemble mucosal-like mast cells, whereas the SCF-derived bone marrow mast cells which resemble connecti ve tissue-like mast cells had a consti tu ti ve expression of IL- 12. Therefore, at least in the murine system, distinct differences do exist between the connective tissue-like mast cell and the mucosal­like mast cell. Mast Cell Growth Factors These two types of murine mast cells exist in two different microenvironments as mentioned before. One factor that contributes to the differences In these microenvironments IS the cytokine makeup of the microenvironment. Connecti ve tissue-like mast cells reside in the connective tissue, such as the skin, where large numbers of fibroblasts also reside (60). Based on the close association of mast cells with fibroblasts, studies were performed to see if fibroblasts alone could support mast cell growth in vitro. Many 16 studies with both mouse and rat mast cells demonstrated that indeed, fibroblast monolayers alone could support mast cell survival (61, 62). Further studies demonstrated that one cytokine expressed by fibroblasts could replace the fibroblast monolayer in supporting the growth of mast cells (63). This cytokine was designated stem cell factor (SCF), which is also called mast cell growth factor (MGF), or c­kit ligand. The latter designation, c-kit ligand, was given, since the cell surface molecule that SCF binds to and ligates is the c-kit receptor. Further studies, based on the observation that mast cells are of a hematopoietic lineage, demonstrated that mast cells with a connecti ve tissue-like phenotype could be generated from bone marrow by culturing mouse bone marrow mast cells in the presence of SCF for 3-4 weeks (64). The other mast cell subclass, mucosal mast cells, resides in the mucosa, which can have a high level of IL-3 production, due to the presence of activated T cells (for review see 65). In fact, it was observed that nude mice, which lack T cells due to the absence of a thymus, have decreased numbers of mucosal-like mast cells in situations of rapid hyperplasia, but normal levels of connective tissue-like mast cells (66). It was later determined that this lack of mucosal-like mast cells in situations of rapid hyperplasia was a result of the lack of IL-3 with the study by Abe et al. (67). In this 17 study, it was observed that athymic nude (nu/nu) mice that were infected with the parasitic nematode Strongyloides ratti were unable to expel the worms and had low levels of mucosal-like mast cells. However, when these mice were repeatedly injected with IL-3, the number of worms was considerably lower, and the number of mucosal-like mast cells significantly increased (67). Further studies observed that IL-3 could drive the differentiation of mast cells from bone marrow, and purified cultures of mast cells could be obtained after 3-4 weeks of culture (68). Clearly, SCF and IL-3 are significant to the differentiation and proliferation of mast cells. However, the similarity between these two growth factors is minimal. SCF is expressed by fibroblasts, keratinocytes, endothelial cells, and adipocytes and is a crucial growth factor in the differentiation and migration of embryonic cells (69, 70). Its receptor, the c-kit tyrosine kinase receptor, is expressed by developing hematopoietic progenitors, primordial germ cells, melanoblasts, erythrocytes, and mast cells (71). Developing lymphocytes that express the c-kit receptor are pro-B, pre-B, and Pro-T cells (71). C -kit receptor expression has also been observed in nerve cells and a small subset of natural killer cells (72). The receptor is expressed as a transmen1brane protein and when bound by its ligand, SCF, will dimerize, leading to enhanced activity of the 18 tyrosine kinase (73). For these developing cells, SCF may also serve as a chemoattractant for embryonic nerve cells, primordial germ cells, and hematopoietic progenitors (74, 75). Interestingly, SCF retains many of these functions for the mast cell. For the mast cell, SCF can serve as growth factor, chemoattractant, and degranulating agent (75, 76). SCF has been demonstrated to be a very potent cytokine!chemokine, since mice that are homozygous knockou ts for SCF do not survive. The only mice that survive with mutations in the SCF gene are mice that are heterozygous. The best example of a mouse that is SCF deficient is the Steel (SL) mouse, SlISld , which IS macrocytic anemIC and sterile and lacks cutaneous melanocytes (77). Similar phenotypes are observed with the white spotted mouse (W), for which at least two alleles, v and s, have been isolated (77). The v allele is a rearrangement of the c-kit receptor locus, and the s allele is a small deletion of the c-kit gene (77). It was noted that these mice exhibited deficiencies in coat color (piebaldism), mental capabilities and other neural associated functions (retardation), erythrocyte numbers (anemia), and reproduction (sterility) (77). However, these mice are not truly mast cell deficient, with mast cells in the white spotted mouse approximately <1 % of the number of mast cells in a wild type mouse, but nevertheless, still present (77). Similarly, with steel mIce, <1 % of the numbers of mast cells was present compared with the con genic normal mice (77). Similarly, IL-3 is a pleiotropic cytokine. Similar to SCF, IL-3 19 IS a growth factor that is important for the proliferation of he­matopoietic cells, including pre-B and pro-B cells, and pro-T lymphocytes (78, 79). Its cellular sources include activated T cells, mast cells, and keratinocytes (65, 80, 81) and normally is not produced in the bone marrow (65). Since all of these cells are found In the periphery, IL-3 was thought to contribute to the proliferation of hematopoietic cells in an emergency. However, a recent study by Nishinakamura et al. indicated that IL-3 probably does not play a unique role in this function (82). In their study, which consisted of generating a mouse that lacked the ~c subunit of the IL-3R (AIC2B), no striking abnormalities associated with IL-3 function deficiency was observed. However, the ~c subunit is also utilized by the IL-5 receptor and the GM-CSF receptor (82). In this family of cytokine receptors (IL-3R, IL-5R, GM-CSFR), the ~c subunit serves as the signaling component, and the alpha subunits serve to confer specificity for the cytokine ligand (83, 84). Thus, a ~c knockout removes some IL-3 and all IL-5 and GM-CSF function. In this particular study, then, no abnormalities were associated with the decreased level of IL-3 receptor function, but abnormalities associated with the lack of IL-5 and GM-CSF were observed. This included a decrease in the number of peripheral eosinophils and al veolar proteinosis-like symptoms. These results would indicate 20 that IL-3 serves a redundant role with other cytokines. Due to the possibility that another p signaling subunit can be utilized by the IL- 3R in the mouse (PIL-3 or AIC2A), a double mutant mouse, pc deficient and an IL-3 deficient, was generated. This effectively removed all IL-3 dependent functions in the mouse. Again, no dramatic abnormalities associated with IL-3 were observed to develop. This would also indicate that IL-3 serves a redundant role and that other cytokines, besides just IL-5 and GM-CSF, can substitute for IL-3 in its absence. In the mouse system, other cytokines that have been demonstrated to increase further mast cell proliferation are IL-4, IL- 9, IL-IO, and IL-15 (6, 85). However, although they do contribute, they are not capable of generating increased proliferation by themselves. IL-15 is similar to IL-2, in that it can serve as a growth factor for T cells (86). It has also been demonstrated to induce a Th 1 phenotype in CD4 + T cells, in that CD4+ T cells incubated with IL-15 will increase their production of IFN-y (87). In contrast, IL-4, IL-9, 21 and IL-I0 have a primarily anti-inflammatory effect on other cell types. For example, IL-4 and IL-I0 are both expressed by the anti­inflammatory type of T helper cell, the Th2 type (for review see 41). IL-4 can contribute to the class switching of antibody by a B cell to the IgE class, can cause the differentiation of T helper cells into Th2 like cells, and can cause the macrophage to downregulate its expressIon of IL-6, IL-l, and TNF-a (for review see 48). IL-IO has similar properties in that it, too, can cause macrophages to downregulate their expression of IL-6, IL-l, and TNF-a production (88). IL-9 is produced by activated T helper clones of the Th2 type and, as such, is categorized as a Th2 type cytokine (89). IL-9 functions primarily as a growth factor for T helper cell lines, myeloid cell lines, and mast cells. One report has demonstrated that it may have the potential for activating T cells. It was observed that a murine T cell line treated with IL-9 had increased levels of transcript for the granzymes A and B. So far, the inflammatory or anti-inflammatory inducing nature of these cytokines as seen with other cell types has not correspondingly been addressed with respect to the mast cell. However, some studies have demonstrated that these cytokines alter the granule content of mast cells. 22 One change In granule content exhibited by mast cells towards these cytokines is a change in protease content. Systematic analyses of the different types of proteases that are expressed by mas t cells in different cytokine milieus have been performed. Eklund et al. demonstrated that IL-3-derived bone marrow mast cells express high steady state levels of transcripts for murine mast cell protease 5 (mMCP5) and that KL treatment of these cells can induce the accumulation of mMCP4 transcripts (92). IL-IO or a combination of IL-9 and KL can induce the accumulation of mMCPl and mMCP2 transcripts. IL-4 suppresses the effects of IL-9 and IL-IO in that accumulation of mMCP 1 and 2 transcripts is inhibited (92). These studies indicate that mast cells have a variety of phenotypes that may not be limited to just a mucosal-like phenotype or connective tissue-like phenotype. Mast cell phenotypes may be a complex set of phenotypes dependent upon the combination of extracellular cytokines present in the microenvironment of the mast cell. Costimulatory Molecules Costimulatory molecules are a group of molecules that were originally identified on the surface of T cells and B cells. These molecules participate in the stimulation process that occurs when B cells present antigen to T cells. They serve primarily to enhance or 23 Increase the stimulation level that is achieved by both the T cell and B cell during antigen presentation. However, there are exceptions. For example, some costimulatory molecules serve to downregulate the stimulatory process. Costimulatory molecules that are expressed by the T cell include CD28, Cytotoxic T Lymphocyte-Associated Molecule-4 Receptor (CTLA-4), 4-1 BB, and CD40 Ligand (CD40L) (93- 97). Costimulatory molecules expressed by the B cell are B7-1 (CD80), B7-2 (CD86), a putative B7-3, 4-1BB Ligand (4-1BBL), and CD40 (98, 99). Costimulatory molecules are also expressed by cells other than T cells and B cells. Those costimulatory molecules that are expressed by T cells and are expressed by other cell types are CD28 [natural killer cells (100)] and CD40L [human vascular endothelial cells, macrophages, and smooth muscle cells (101)]. Those costimulatory molecules that are expressed by B cells and are expressed by other cell types are B7-1 and B7-2 [activated monocytes, dendritic cells, and activated T cells (102)], CD40 [B cells, rnonocytes, dendritic cells, endothelial cells, and fibroblasts (103)],· and 4-1BBL [splenic dendritic cells (104)]. The expression level of these costimulatory molecules vanes with cell type and activation status. CD28 is expressed constitutively by T cells and NK-1.1 + cells, and its expression level can be increased at the surface of the T cells with activation (100, 105). As of yet, 4- 1BB and CTLA-4 have only been observed to be expressed by activated T cells, with CTLA-4 expressed at higher levels on CD8+ cells than CD4+ cells (106). With CD40L, human endothelial cells, smooth muscle cells, and macrophages have a low constitutive expression level, but with IL-1~, TNF-a, or IFN-y treatment the cell 24 surface expression of CD40L can be increased (101). 4-1BBL is expressed on activated B cells and activated macrophages (104, 107). B7-1 and B7-2 are expressed by antigen presenting cells and can be induced with the ligation of CD40 at the surface of the antigen presenting cells (108). B7-1 and B7-2 have also been observed to be expressed by human endothelial cells, with B7-2 expressed 24 hr after stimulation, and B7-1 expressed 72 hr after stimulation (108). This pattern of expression, where B7 -2 expression is increased first and B7-1 expression increased later, was also seen with human B cells that were activated. Again, B7-2 expression was observed to be greatly increased within 24 hr after activation, and B7-1 was observed to be expressed 2-3 days later (99, 102). Observations such as these have led to the theory that the principle ligand for CD28 is B7-2 and the principal ligand for CTLA-4 is B7-1 (102). Both B7-1 and B7-2 have also been observed to be expressed by activated T cells (109, 110). With CD40, constitutive expression has been 25 observed with B cells, monocytes, dendritic cells, and fibroblasts (103) . Costimulation VIa costimulatory molecules is based on the interaction of these molecules expressed by T cells and B cells. CD28 on the T cell, when ligated by B7-1 or B7-2, can lead to, among other consequences, increased IL-2, IFN-,)" GM-CSF, Lymphotoxin, IL-3, IL- 4, IL-5, or TNF-a production by T cells, due to either or both mRNA stabilization or increased transcription (111-114). These cytokines, especially IL-2, will lead to the further growth and proliferation of the T cells in both a paracrine and autocrine fashion. CTLA-4 also binds to B7-1 and B7-2, and binds with 10- to 20-fold greater affinity than CD28 for either of these molecules (98). However, CTLA-4 is an exception in the costimulatory molecule group, in that the signal derived from CTLA-4 does not result in the increase of T cell proliferation, due to the absence of IL-2 production. Ligation of CTLA-4 may actually lead to the inhibition of IL-2 accumulation. CD40L is similar to CTLA-4 in that ligation of CD40L also does not appear to lead to the increased proliferation of the T cell (116). For the B cell, however, ligation of CD40 at the surface of the B cell by CD40L at the surface of an activated T cell will result in the class switching of antibody to IgE by the B cell, increased levels of B7-1 26 and B7-2 expressIon, increased proliferation of the B cell, and increased expression of 4-1 BBL by the B cell (104). Interestingly, 4- 1BB on the activated T cell can serve as a costimulatory molecule in the absence of CD28, resulting in a strong proliferative response and cytokine production by the T cells when 4-1 BB is ligated (104, 117). More insight into the functions of these costimulatory molecules was achieved with the production of mice where the gene for the costimulatory molecule was knocked out. So far, knockout mice have been generated for CD28, CTLA-4, B 7 -1, CD40, and CD40L. The knockout mouse for B7-1 did not display any critical reduction in the immune response. No reduction in the number of T cells or B cells was observed, nor any reduction in the concentration of serum immunoglobulins (118). These B7-1 deficient mice also respond normally to mitogens (118). These data indicated a second ligand for CD28 or CTLA-4 and, in fact, led to the isolation and cloning of B7-2 by Freeman et al. (118). As of yet, a double knockou t for B 7 -1 and B7-2 has not been generated to test the hypothesis that a putative' B7-3 exists (99). For the CD40 knockout, the phenotype was more dramatic. B cells from CD40 deficient mice were unable to function as antigen presenting cells. However, B cells from CD40 deficient mice, once treated with LPS, which upregulates the expression of B7 molecules on B cells, were able to present antigen and stimulate an 27 alloresponse. Similarly, mIce that are CD40L deficient do not form germinal centers in response to thymus dependent humoral immune responses, nor do they produce antigen specific IgG 1 or elevate IgM levels in response to thymus dependent antigens. Thus, one critical CD40 function, which was illuminated with the CD40 deficient mice, is to stimulate B cells and increase the expression of both B7 molecules on B cells (119). For the CD28 deficient mice, very little was observed in the way of a dramatic altered phenotype. The development of T and B cells was normal (120). Immunoglobulin production was 20% of normal, and the antibody class that was most reduced was IgG2a (120). As was expected, B7 -dependent functions, such as antigen presentation to T helper cells were inhibited (121). However, most other T cell functions were unaffected, such as cytolytic functions. This absence of a dramatic deficiency in T cell function led to the development of a CTLA-4 knockout mouse. This, surprisingly, of all the costimulatory molecules addressed so far, had the most dramatic phenotype. CTLA-4 deficient mice had an increased level of lymphoproliferation, resulting in fatal multiorgan. failure (122). These mice exhibited severe myocarditis and pancreatitis and died within 3-4 weeks of age. Most interesting was the observation that the CTLA-4 deficient mIce had a fivefold increase in the number of CD4-CD8- T cells in the thyn1us along with a 28 10-fold decrease in CD4+CD8+ T cells in the thymus. This indicated a critical negative role for CTLA-4 in the costimulatory process, and may indicate a role in thymocyte maturation for CTLA-4. Also observed was an increase in the number of total CD3+ T cells in the spleen and the lymph nodes, possibly indicating a role for CTLA-4 in the regulation of peripheral T cells (122). CD28 Signaling Pathway Of the costimulatory molecules expressed by T cells, CD28 IS the best characterized with respect to cell signaling. The CD28 cell signaling pathway has been analyzed primarily in human T cell lines, such as lurkat T cells. CD28 is expressed constitutively at the cell surface on CD4+ T helper cells and approximately 50% of CD8+ T cells (123). The expression level of cell surface CD28 on T cells can be increased with activation with PMA or anti-CD3 and will peak at about 48 hours after the activation event (105), Once crosslinked, CD28 then activates various phosphotyrosine kinases at the cell membrane, including phosphatidylinositol 3' -kinase (PI3 kinase) (124). This results in the phosphorylation of CD28 and various downstream kinases, such as MAPK and SAPK. These downstream kinases phosphorylate transcription factors such as c-jun. The activated c-jun binds to c-fos, forming the transcription factor AP-l. 29 AP-1 binds to the nuclear factor, NF-AT, that is activated through the ligation of the T cell receptor. The NF-A T / AP-1 complex can then bind to the CD28 response element (CD28RE) in various genes. NF-KB can also bind to the CD28RE. Some genes that have the CD28RE in their promoter region are IL-2, GM-CSF, IFN-y, IL-3, and human IL-8 (125). These increases in cytokine expression are a result of both TCR ligation and CD28 ligation, since NF-A T is provided by the ligation of the TCR, and AP-1 ligation is provided by the ligation of CD28 (124). Thus, in the absence of TCR ligation, CD28 crosslinking alone will lead to processes that do not have NF-AT involved. An example of some events that occur with CD28 ligation in the absence of TCR ligation IS the activation of c-jun, but not c-fos. Thus, in this situation CD28 crosslinking does not lead to increased IL-2 production or other cytokine production, so no proliferation of the T cell is observed. Other cytokines that are increased after CD28 crosslinking but may or may not have CD28RE in their promoters are IL-13, IL-4, and IL-5 (127, 128). The end result is that CD28 crosslinking simultaneously with TCR crosslinking leads to the increased production of these cytokines. 30 IL-13 IL-13 was originally isolated in a screen looking for novel cytokines that are expressed by activated human T cells (127). IL-13 is very similar in function to IL-4, in that it can cause B cell proliferation, increased CD23 expression, and antibody class switching to IgE (129, 130). Also similar to IL-4 and other Th2 type cytokines, IL-13 can downregulate the production of IL-6 by peripheral blood mononuclear cells and the accumulation of mRNA for IL-l p and TNF-a. (127). However, in contrast to IL-4, T cells that are cultured in the presence of IL-13 do not develop into Th2 like cells (131). Introduction to Work in This Thesis Material presented in this thesis addresses two means by which the mast cell can "sense" its microenvironments, cytokine uptake, and costimulatory molecule ligation. Specifically, the effect of IL-3 on the expression of cytokines by mast cells was analyzed, . and the expression of CD28 and its effect on cytokine expression by . the mast cell was analyzed. These studies were based on the prenlise that the composition of mast cell granule components can be changed in different situations or microenvironments. 31 An indication of IL-3 altering the cytokine expression of mast cells comes from the observation that bone marrow cells grown in the presence of IL-3 for 3-4 weeks are phenotypically similar to mucosal-like mast cells. Direct support for IL-3 affecting cytokine expression by mast cells came from the observation by Smith et al. that IL-3-derived bone marrow mast cells express IL-4 transcripts constitutively, but SCF-derived bone marrow mast cells do not (39). Extending this observation, the expression of other anti­inflammatory cytokines by IL-3-derived mast cells was addressed, as well as the effect of IL-3 treatment upon SCF-derived bone marrow-derived mast cells. Transcript analysis via RT -RPCR determined that IL-3-derived bone marrow mast cells do have constitutive levels of transcripts for two anti-inflammatory cytokines, IL-IO and IL-13. FACscan analysis using intracellular staining demonstrated that IL-3-derived bone marrow mast cells express IL-IO protein constitutively, presumably storing it in the granules. Also observed was the production of IL-IO by SCF-derived mast cells 4 days after IL-3 treatment. The expression of costimulatory molecules was proposed when it was observed that mast cells can present antigen via MHC Class II to T cells (32). Although the expression of B7-1 and B7-2 was not addressed specifically, their expression by mast cells is possible, 32 SInce successful antigen presentation via MHC Class II requIres the presence of a costimulatory molecule, such as B7-1 or B7-2. FACscan analysis determined that IL-3-derived bone marrow mast cells express CD28 constitutively at low levels, and SCF-derived bone marrow cells can be made to increase their expression of CD28 by treatment with PMA, LPS, or the bacterial lipoprotein OspA. Also, mast cell CD28 was found to share similar signaling components with T cell CD28, since with both cell types, CD28 crosslinking led to increases in the c-jun transcript levels and IL-13 transcript levels as determined by transcript analysis via RT -RPCR. 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USA 90:3730. 131. de Vries, J.E., and G. Zurawski. 1995. Immunoregulatory properties of IL-13: its potential role in atopic disease. Int. Arch. Allergy Immun. 106: 175. 47 CHAPTER II BONE MARROW-DERIVED MAST CELLS 49 Abstract In order to conduct the studies that are presented later in this thesis, it was necessary to generate a large number of mast cells. Because the yield of purified, homogeneous mast cells from the peritoneum, or mucosal regions, including the intestine, is very low, bone marrow-derived mast cells were used. These mast cells have been previously denlonstrated to provide larger numbers of cells that are phenotypically similar to in vivo derived mature mast cells (1). Previous studies on bone marrow-derived mast cells used fibroblast mono layers and recornbinant rat SCF as sources of SCF, and WEHI-3 cell supernatant as a source of IL-3. The similarities between bone marrow-derived mast cells and in vivo derived mature mast cells include the production of histamine, the production of various proteases, the production of proteoglycans, and the ability to degranulate in response to antigen bound IgE (1). Bone marrow mast cells generated in this work were generated with recombinant mouse SCF and recombinant mouse IL-3. Via FACscan analysis, these SCF-derived bone marrow mast cells were determined to express c-kit and IgE receptors, both of which are mast cell markers. Via degranulation assays that analyzed the release of p-glucuronidase, it was determined that both the SCF and IL-3-derived 50 bone marrow mast cells were capable of degranulating in response to treatment with IgE and antigen. These results indicate, then, that using supernatant from mIL-3 producing 653 myeloma cells and supernatant from mSCF producing CHO cells with murine bone marrow cultures yields mast cells. Introduction Generating mast cells from mouse bone marrow requtres culturing mouse bone marrow cells in the presence of either SCF or IL-3 for at least 3 weeks. These two models were originally based on the observations that the nude mouse has a decreased level of mucosal-like mast cells and that fibroblasts contribute to the growth and proliferation of mast cells (2, 3). Other cytokines/growth factors besides IL-3 and kit ligand have been observed to contribute to increased proliferation of bone marrow-derived mast cells, including IL-4, IL-9, and IL-IO (4). Our specific system used IL-3 and SCF that was deri ved from transformed cell lines that had been transfected' with cDNA for mouse IL-3 or mouse SCF. This is in contrast to culturing the bone marrow cells in the presence of cells that normally express SCF (fibroblast monolayers) or IL-3 (activated T cells or keratinocytes) (5, 6). Specifically, we used supernatant from a 653 myeloma cell line that had been transfected with mouse IL-3 51 cDNA as the source of IL-3, and supernatant from a Chinese Hamster Ovary cell line that had been transfected with the mouse SCF cDNA as a source of SCF (7). Supernatant from these cells were used for the bone marrow culture in a concentration of 10% for both the SCF and IL-3-derived mast cells. Again, this is in contrast to some other studies that use Concanavalin A (ConA)-stimulated splenocytes (6) as a source of IL-3, WEHI-3 cell lines as their source of IL-3 (8), or fibroblast monolayers as their source of SCF. By using the 653 and CHO cell lines, the number of cytokines in the culture was restricted, and a large number of phenotypically similar mast cells could be generated. In contrast, the WEHI-3 cell line is a myelomonocytic leukemia cell line that had been obtained by injecting paraffin oil and testosterone into a Balb/c mouse. The mouse developed a tumor In the first 6 months of age, from which the WEHI-3 (Walter and Eliza Hall Institute of Medical Research) cell lines are derived (9). Later, Lee et al. demonstrated that the WEHI-3 cell lines constitutively express, among other cytokines, IL-3 (10). This is in contrast to the.· 653 myeloma cell line, which as a result of a transfection with murine IL-3 cDN A, produces predominantly IL-3 (1 J.lg/ml per 106 cells, 24 hr) and some IL-IO (100 ng/ml per 106 cells, 24 hr). 52 Similarly, a fibroblast monolayer would express other cytokines and growth factors other than SCF and so would also not provide a well-defined means of generating mast cells. Because it IS known that other cytokines can have an effect on the growth and proliferation of mast cells, our reasoning from the outset was to restrict the number of variables (cytokines) and analyze the specific effects that one or two cytokines had on the function of mast cells. Other groups have generated data that mast cells derived from bone marrow USIng the WEHI-3b cell line, or fibroblast monolayers, were phenotypically similar to either mucosal-like mast cells or connective tissue-like mast cells (11, 12). However, as we were using different sources of recombinant SCF and IL-3, we needed to demonstrate that our specific models were also generating mast cells. In order to do this, we used various mast cell markers, such as the c­kit receptor and IgE receptor to phenotype our bone marrow-derived cells and demonstrate that they were mast cells. Two enzymatic studies analyzing the extent of degranulation in response to high affinity FCERI crosslinking were used to indicate that the cells generated were functioning mast cells. 53 Results As no antibody has been generated that recognizes the mouse high affinity IgE receptor, a different method for detecting IgE receptors has been used. This method was first used by Rottem et al. (13). This consists of detecting IgE bound to the surface of the cell. Rottem's approach was to incubate mast cells with FITC-Iabeled IgE. A modification of this was the approach of Lantz et al. This consisted of incubating mast cells with IgE first and then staining for the presence of IgE bound to the surface of the cell by using anti-mouse IgE antisera, followed by FACS analysis (14). This does not differentiate between the high affinity IgE receptor versus the low affinity IgE receptor (CD23) but does at least demonstrate that IgE is being bound by the n1ast cell. As demonstrated in Figure 2.1, bone marrow cells that were cultured for 7 weeks in the presence of supernatant from the IL-3 producing 653 myeloma cell line express c-kit and IgE receptors. As demonstrated in Figure 2.2, bone marrow cells cultured for 14 days in the presence of supernatant from the IL-3 producing 653 cell line also express IgE receptors and c-kit receptors. However, at this stage of development, there appears to be a small subpopulation of c-kir cells still present. Figures 2.3 and 2.4 demonstrate that bone Figure 2.1 FACscan analysis of c-kit and IgE receptor expression by 7-week IL-3-derived mouse bone marrow cells. Mouse bone marrow cells that had been cultured for 7 weeks in the presence of supernatant from mIL-3 transfected 653 myeloma cells were analyzed via FACS analysis for c-kit and IgE receptors. FITC conjugated rat IgG2b is the isotype control for the FITC conjugated Rat anti-Mouse c-kit antisera (clone 2BS). Biotinylated rat IgG 1 is the isotype control for biotinylated Rat anti-Mouse IgE antisera. 54 55 0 0 co co ,0.. ... ,0... .. 0 anti-c-kit 0 CD CD 0 I 0 UJLO UJLO Eo Eo :;,.q- :;,.q- 0°('01) 0°('01) 0 0 C\I C\I 0 0 T""" T""" 103 104 104 Figure 2.2 FACscan analysis of c-kit and IgE receptor expression by 2-week IL-3-deri ved mouse bone marrow cells. Mouse bone marrow cells that had been cultured for 14 days in the presence of supernatant from mIL-3 transfected 653 myeloma cells were analyzed via F ACS analysis for c-kit and IgE receptors. FITC conjugated rat IgG2b is the isotype control for the FITC conjugated Rat anti-Mouse c-kit antisera (clone 2BS). Biotinylated rat IgG 1 is the isotype control for biotinylated Rat anti-Mouse IgE antisera. 56 57 0 0 co co t0- - 0 IgG1 IgG2b t-- 0 0 1 co ',I co 0 0 001.0 001.0 co anti-c-kit co :Jv :Jv anti-lgE 0 0 (JO (JO ('t) ('t) 0 0 C\I C\I .0. - .0. - 58 Figure 2.3 FACscan analysis of c-kit and IgE receptor expression by 7 -week SCF-deri ved mouse bone marrow cells. Mouse bone marrow cells that had been cultured for 7 weeks in the presence of supernatant from mSCF transfected CHO cells were analyzed via FACS analysis for c-kit and IgE receptors. FITC conjugated rat IgG2a is the isotype control for FITC conjugated Rat anti-Mouse c-kit antisera (clone ACK-4). Biotinylated rat IgG 1 is the isotype control for biotinylated Rat anti-Mouse IgE antisera. 59 0 0 0 0 T""" T""" 0 IgG2a 0 co co -(cc/ )o0 --,/ anti-c-kit -(cc/ )o0 :J :J anti-lgE 00 , 00 ()~ ()~ 0 0 / C\I C\I 103 104 104 60 Figure 2.4 FACscan analysis of c-kit and IgE receptor expression by 3-week SCF-derived mouse bone marrow cells. Mouse bone marrow cells that had been cultured for 19 days in the presence of supernatant from mSCF transfected CHO cells were analyzed via FACS analysis for c-kit and IgE receptors. FITC conjugated rat IgG2a is the isotype control for FITC conjugated Rat anti-Mouse c-kit antisera (clone ACK-4). Biotinylated rat IgG 1 is the isotype control for biotinylated Rat anti-Mouse IgE antisera. 61 0 0 0,. .. ,0.. . 0 0 a:> a:> IgG1 2lg anti-c-kit 2lg ,/ anti-lgE c: ::I c: 00 ::I ¥ ()v i 00 ()v 0 0 C\I C\I 104 104 62 marrow mast cells cultured for 7 weeks or 19 days in the presence of supernatant from the SCF producing CHO cell line also express the c­kit receptor and an IgE receptor. In order to address the functionality of the IgE receptor that was identified as being expressed at the surface of these bone marrow mast cells, we conducted various analyses to examIne the degranulation properties of these cells. Briefly, these cells were incubated with IgE specific against dinitrophenyl and then treated with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) for 45 mIn. Degranulation was analyzed by examining the quantity of the release of two preformed enzymes into the supernatant. These two enzymes are ~-hexosaminidase and ~-glucuronidase, both of which are lysosomal enzymes. By measuring the release of these lysosomal enzymes, the amount of degranulation by the mast cell can be measured. Enzyme release can be measured by adding chromogenic substrates for these enzymes to the supernatant of the degranulated nlast cell and measuring the activity of the enzyme. For the ~- hexosaminidase, the substrate used was p-Nitrophenyl N-Acetyl-~­Glucosaminide, and for the p-glucuronidase, the substrate used was phenolphthalein glucuronic acid (15). In Figure 2.5, bone marrow-derived cells that had been cultured for 8-9 weeks in the presence of supernatant from IL-3 producing 653 myeloma cells were assayed for degranulation in response to IgE and antigen. Thirty percent release of total P -G I u- 63 curonidase activity occurred with the addition of IgE+ antigen, with background levels of about 5% for p-Glucuronidase release from cells treated with only DNP or only IgE. Similar results were obtained with the p-Hexosaminidase assay. In Figure 2.6, bone marrow-derived mast cells that had been cultured in SCF for 7 weeks degran­ulated with the addition of IgE and antigen. The percentage of P-glucuronidase release was determined by measuring the fluorescence at 540 nm. Again, similar to the MMC, the CTMC provided about a 30% release of p-Glucuronidase activity. However, background levels of release are twice as high as the MMC. This may be due to the degranulating ability of SCF, since decreasing the concentration of IgE below 1 j.lg/ml did not reduce the background level of degranulatio.n. Sitnilar results were also obtained with the p-hexosaminidase assay. ~ As demonstrated in Figures 2.1-2.6, both types of bone marrow-derived cells do express c-kit and IgE receptors and degranulate in response to IgE and antigen. However, the SCF­derived bone marrow mast cells require a higher concentration of 64 Figure 2.5 Degranulation by IL-3-derived mouse bone marrow cells in response to IgE + antigen. Mouse bone marrow cells that had been cultured for 8-9 weeks in the presence of supernatant from the IL-3 producing 653 cell line were treated with JgE + antigen. Degranulation was measured via the release of p-Glucuronidase. Fluorescence was measured at 540 nm. ..~ ................. ...,... ............................... ...,... .............................. ..'.." ................ ...,.. ................................ ..,... ....'." ................................................ .II...' ............ ."..... .......... ........... .. . ~ JgE 50ng/mt + DNP - f~rrrtrrmtttff~(}j~t[ -; 7m IgE 50 nglml - }?it I ~ ..................... .".... ........................ . DNP alone - j}![tt ---1 ::::::: No IgFJNo DNP - m~j ~ ..:..:.:.:.::.: o I 10 I 20 I 30 % Release/Glucuronidase 40 r.:::l I,;.:.;.:J 65 66 Figure 2.6 Degranulation by SCF-derived mouse bone marrow cells in response to IgE + antigen. Mouse bone marrow cells that had been cultured for 6 weeks in the presence of supernatant from SCF producing CHO cells were treated with IgE + antigen. Degranulation was measured via the release of p-Glucuronidase. Fluorescence was measured at 540 nm. ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: IgE 1000ng/ml + DNP - ~~{{}{??????{???????t~ -i ::::::::::::::::::: r gE 1000ng/ml alone - ~~~~l~l~(~~t~~ :::::::::::::::::::::: DNP alone - ~)~~~~~tm ~ ::::::::::::::::::::::::::::::::::::: No IgFJNo DNP - }~{{:}})~:~:~:}~ ~ ::::::::::::::::::::::::::::::::::::: o I 10 I 20 I 30 % Release/Glucuronidase 40 r.:::l ~ 67 68 IgE for degranulation with antigen than the IL-3-derived bone marrow mast cells. Both concentrations though, 50 ng/ml for the IL- 3-derived bone marrow cells and 1000 ng/ml for the SCF-derived bone marrow cells are concentrations that are either the same concentration or lower than concentrations used by other researchers for mast cells. Discussion These data indicate, then, that these two cell types, in fact, express IgE receptors. These data also indicate that since the release of the two enzymes by both cell types is dependent upon the crosslinking of the IgE against specific antigen, a portion of these IgE receptors are the high affinity IgE receptors (Fc£RI). Also, since these cells express the mast cell marker, c-kit, these cells are not basophils but are mast cells. Further characterization of these cells will be presented in Chapters III and IV, such as the lack of IL-2R transcripts. Together these data indicate that these two cell types are indeed mast cells and not basophils, natural killer cells, T cells, B cells, or macrophages. Therefore, using these two different types of bone marrow­derived mast cells, important questions that will be addressed are 69 the following: Are these mast cell types capable of responding to cytokines by changing their cytokine profile? Do these cell types express costimulatory molecules, and what signals might the ligation of these molecules provide for the mast cell? Last, do these two types of mast cells respond differently to the same stimuli? Experimental Procedures Cells and Tissue Culture The 653 cell line transfected with mouse IL-3 eDNA was obtained from Ray Daynes. The CHO cell line that was transfected with mouse SCF eDNA sequence was obtained from Genetics Institute, Cambridge, Massachusetts. Bone marrow was flushed from the tibia and femurs of female outbred mice. The bone marrow was then cultured in 90% RPMI and 10% supernatant from either the 653 cells or the CHO cells. Culture times were for 19 days, 6 weeks, or 7 weeks for the SCF-derived bone marrow mast cells and for 14 days, 7 weeks, or 8-9 weeks for the IL-3-derived bone marrow mast cells. Mouse serum was prepared from cardiac punctures. F ACS Analysis For the analysis of IgE receptors, mast cells were incubated with mouse IgE (monoclonal IgE against DNP-BSA) for 30 min in RPMI at 37°C. Cells were then washed and stained with Rat anti- Mouse IgE antisera In the presence of mouse serum followed by s trepta vi di n-p h ycoerythrin. 70 Antisera used were FITC conjugated anti-c-kit (clone 2B8 from Pharmingen cat# 01904D) and FITC conjugated anti-c-kit (clone ACK- 4 from University of Utah Stem Cell Core Facility). Isotype controls used were FITC conjugated IgG2b (from Pharmingen cat# 11034C) and FITC conjugated Rat IgG2a (from Pharmingen cat# 11024C). Biotinylated Rat anti-Mouse IgE antisera was from Pharmingen (cat# 02132D). Isotype control was biotinylated rat IgG 1, obtained from Phanningen cat# 110 12C. Streptavidin-phycoerythrin was obtained from Pharmingen (cat# 13025D). Degranulation Assay Mast cells were incubated with 50 ng/ml IgE (IL-3-derived bone marrow mast cells) or 1 J.1g/ml IgE (SCF-derived bone marrow mast cells) for at least 30 min in RPMI at 37°C. Cells were then washed and resuspended in 1 ml 1 X Pipes buffer with 100 ng/ml DNP-BSA. Cells were then plated onto BSA coated 6 well dishes for at least 45 min. BSA coated 6 well dishes were prepared by adding 1-5 ml of IX Pipes, 1 % BSA for at least 30 min with consequent removal 71 before the addition of cells. Supernatant was then collected from the mast cells incubated in the BSA coated 6 well dishes. Cell pellets were resuspended in 1 ml of 1 X Pipes and sonicated. Two hundred microliters of supernatant or sonicate was added to 50 ul of 0.01 M phenolphthalein glucuronic acid and 50 ul O.IM Sodium Acetate buffer pH 4.5 and 200 ul of 1 X Pipes. Samples were incubated overnight at 37°C, after which 500 ul of 0.4M Glycine Stop Solution pH 10 was added. The percentage of release was determined by the following equation: 0.0. supernatant % Release = (0.0. supernatant + 0.0. cell pellet) Fluorescence was measured at 540 nm on a spectrophotometer (15). Phenolphthalein glucuronic acid was obtained from Sigma (cat# P- 0501). Mouse IgE specific against DNP was obtained from Sigma (cat# D-8406). DNP-BSA was obtained from Molecular Probes (cat# A843). ~-Hexosaminidase assay was conducted similarly. The substrate used was O.OIM p-Nitrophenyl N-Acetyl-~-Glucosaminide (Sigma cat# N-9376), with 50 ul added to the enzymatic assay in place of the phenolphthalein glucuronic acid. Fluorescence was measured at 400 nm on a spectrophotometer. 72 References 1. Gurish, M.F., and K.F. Austen. 1989. Different mast cell mediators produced by different mast cell phenotypes. C i ba Found. Symp. 147:36. 73 2. Abe, T., and Y. Nawa. 1988. Worm expulsion and mucosal mast cell response induced by repetitive IL-3 administration in Strongyloides ratti-infected nude mice. Immunology 63: 181. 3. Nocka, K., J. Buck, E. Levi, and P. Besmer. 1990. Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors. EMBO 1.9:3287. 4. Denburg, J .A. 1992. Basophil and mast cell lineages In Vitro and In Vivo. Blood 79:846. 5. Razin, E., K.B. Leslie, and J.W. Schrader. 1991. Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. 1. Immllnol. 146:981. 60 Frandji, Po, C. Oskeritzian, F. Cacaraci, J. Lapeyre, R. Peronet, B. David, Jo Guillet, and S. Mecheri. 1993. Antigen-dependent stimulation by bone marrow-derived mast cells of MHC Class II-restricted T cell hybridoma. 1. Inzmunol. 151 :6318. 7. Karasuyama, H., and F. Melchers. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin-2,-3, -4, or -5, using modified eDNA expression vectors. Eur. 1. Inunllnol. 18:97. 8. Malaviya, R., N.J. Twesten, E.A. Ross, S.N. Abraham, and J.D. Pfeifer. 1996. Mast cells process bacterial ags through a phagocytic route for Class I MHC presentation to T cells. 1. Imlnunol. 156: 1490. 9. Warner, N.L., M.A.S. Moore, and D. Metcalf. 1969. A transplantable myelomonocytic leukemia in BALB/c mice: cytology, karyotype, and muramidase content. 1. Nat. Cancer Inst.43:963. 74 10. Lee, J.C., A.J. Hapel, and J.N. Ihle. 1982. Constitutive production of a unique lymphokine (IL-3) by the WEHI-3 cell line. 1. Immunol. 128: 2393. 11. Razin, E., J.N. Ihle, D. Seldin, J.M. Mencia-Huerta, H.R. Katz, P.A. LeBlanc, A. Hein, J.P. Caulfield, K.F. Austen, and R.L. Stevens. 1984. Interleukin-3: a differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan. 1. Immunol. 132: 1479. 12. Levi-Schaffer, F., K.F. Austen, P.M. Gravellese, and R.L. Stevens. 1986. Coculture of interleukin-3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the cells. Proc. Nat!. Acad. Sci. USA 83:6485. 13. Rottem, M., J.P. Goff, J.P. Albert, and D.O. Metcalfe. 1993. The effects of stem cell factor on the ultrastructure of Fc epsilon RI + cells developing in IL-3-dependent murine bone marrow­derived cell cultures. 1. Immunol. 151 :4950. 14. Lantz, C.S., and T.F. Huff. 1995. Murine Kit+ Lineage- bone marrow progenitors express FcrRI! but do not express FceR I until mast cell granule formation. 1. [mlnunol. 154:355. 15. Hohman, R.J. 1993. Measuring degranulation of mast cells. In Current Protocols in bnmunology. J .E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds. John Wiley & Sons, Inc., New York, pp. 7.26.1-7. CHAPTER III MODULATION OF EXPRESSION OF THE ANTI-INFLAMMATORY CYTOKINES INTERLEUKIN-13 AND INTERLEUKIN-I0 BY INTERLEUKIN-3 Reprinted from Eur. 1. Immunol. (1996) 26:49 76 Eur. J. Immunol. 1996. 26: ~9-56 IL-3 modulates the expression of fL-l3 and lL-lO Eric V. Marietta, Yivou Chen and John H. Weis Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City. USA Modulation of expression of the anti-inflammatory cytokines interleukin-13 and interieukin-lO by interleukin-3 Interleukin (IL)-4, IL-lO and IL-13 are cytokines with potent anti-intlammatory activities. Prevention of pathological intlammation at mucosal surfaces appears to be due. in part, to the presence of these cytokines. One potential source for these cytokines is the mast cell which resides at mucosal surfaces. Demonstrated in this report are the findings that bone marrow-derived mucosal-like mast cells constitutively expressed IL-13 whereas bone marrow-derived connective tissue­like mast cells demonstrated IL-13 transcription only after FceRI-mediated acti­vation or the addition of exogenous IL-3. A similar pattern of expression of IL- 10 by these mast cell types was also evident and matches that of IL-4 previously reported. Intracellular cytokine staining indicated that IL-lO protein is constitu­tively expressed by the bone marrow-derived mucosal-like mast cells but is only evident in the bone marrow-derived connective tissue-like mast cells after induc­tion with IL-3. The increase of IL-13 and IL-lO transcripts in the connective tissue-like mast cells following IL-3 treatment is not mast cell specific. in that splenic and bone marrow cells also demonstrated the same phenomenon. These data suggest that mucosal mast cells may have a constitutive repertoire of Th2 cytokines with potential anti-intlammatory activity, while connective tissue mast cells may not. However, production of such cytokines can be induced in the con­nective tissue mast cell and other cell types of the immune response by the addi­tion of IL-3. 1 Introduction The intcrleukins are a class of cytokines that are produced by a variety of cell types. The production of most interleu­kins is dependent upon the activation of specific cells via a variety of induction pathways. These pathways include rcceptor mediated activation via specific ligand binding which can lead to increased proliferation and differentia­tion, and the production of other cytokines [11. The response of a specific cell to defined stimuli can cither be a pre-programmed response or a response influenced by the specific micro-environment in which the cell is found. The latter case has been invoked to explain the development of the Thl (IFN-y and IL-2 producing) T cells and the Th2 (IL-4 and IL-5 producing) T cells from the uncommitted precursor ThO cell during T cell activation (for review see [21). A subset of cytokines known for their anti-inflammatory effects arc IL-4. IL-lO and IL-13. Collectively, they are known to enhance the humoral immune response and sup­press the cell-mediated response. 11·4 can affect prolifera- [I 13871] Correspondence: John H. Weis. Division of Cell Biology and Immunology. Department of Pathology. 50 N. Medical Drive. University of Utah School of Medicine. Salt Lake City. UT84132. USA (Fax: + 1 XO 15 81-4517) Abbreviations: BM·MMC: Bone marrow-derived mucosal mast cell BM-CTMC: Bone marrow connective tissue mast cell Kl: c-kit ligand (also called stem cell factor and mast cell growth fac­tor) RT-RI'CR: Reverse transcriptase-rapid polymcnlse chain reaction Key words: Interleukin- t3 / Interleukin-3 / Interieukin-lO / Mast cell / Inflammation © VCH Verlagsgcsellschaft mhH, D-(i9~5l Wcinheim. 1l}!)6 tion and differentiation of lymphoid cells, intluence class switching of activated B lymphocytes to the IgO 1 and IgE isotypes, and is instrumental in the generation of the Th2 phenotype ofCD4+ helperT cells. In addition. IL-4 acts to suppress the cytotoxic functions of monocytes and mac­rophages by down-regulating the production of the pro­intlammatory cytokines and inhibiting IFN-y-mediatcd activation (for reviews see [3. 4]). I L-lO is a product of activated Th2 cells. Ly·I B cells, mac­rophages, thymocytes and keratinocytes (for review see [5]). Similar to lL-4, IL-lO can promote the proliferation and differentiation of cells of the immune response. IL-W plus anti-CD40 antibodies stimulate human B cells to differentiate into plasma cells but. unlike IL-4. IL-1O has no effect upon isotype switching. Similar to IL-4, IL-1O has potent immunosuppressive activity upon macrophages. This suppression includes the prevention of nitric oxide synthesis following IFN-y stimulation and the inhibition of the production of the intlammatory cytokines IL- I, IL-6 and TNF-u. Additionally. IL·W can help down-regulate the Th 1 response. Interestingly, while mice deficient in IL- 4 appear for the most part to be normal. aside from differ­ences in immunoglobulin isotypes [6]. mice deficient in IL­lO demonstrate a chronic enterocolitis [7]. IL-13 is structurally and functionally similar to IL-4 (for review see [8]). It was first identified as a product of human T cells after activation of peripheral blood mono­nuclear cells via CD28 and TCR cross-linking [9]. The homologous sequence in the mouse was similarly defined as a Th2 cell product requiring T cell activation (Con A) for expression [10]. Both IL-I3 and IL-4 arc found on human chromosome 5 and murine chromosome II in a cluster of genes encoding cytokines that have similar func­tions (IL-5 and granulocyte/macrophage (OM)-CSF). The 00l4-2980196fOIOl·49$IO.IlO + .25fO 50 E. V. Marietta et al. primary functions of IL-13 have been described as anti· inflammatory, in that IL·l3. similar to IL-4 and IL-W, reduces the production of IL-t IL-6. TNF-a and other cytokines by activated macrophages [ll]. Like IL-4, IL-l3 has been implicated in B cell isotype switching to the IgE isotype [12]. IL-l3 has been described as a product of acti­vated T cells and activated mast cells [9. 13], while IL-4 has been described as a product of T cells, mast cells and basophils (14J. The mast cell is capahle of producing a variety of cyto­kines, including IL-l. IL-4. IL-5, IL-12, GM-CSF, TGF·~, and TNF·a (for reviews see [14, 15]). In a fashion analog­ous to that of ThO cells developing Thl or Th2 cytokine profiles, the cytokine potential of the mast cell is influ· enced by its environment. For example, connective tissue­like mast cells derived from bone marrow (BM·CTMC) in the presence of c-kit ligand (KL, also called stem cell fac­tor and mast cell growth factor) constitutively transcribe the genes encoding the IL-12 subunits, but not IL-4. whereas mucosal-like mast cells (MMC) derived from bone marrow in the presence of fL·3 (BM·MMC) tran­scribe IL-4, hut not IL-12 [16]. Cytokine release by these two populations of mast cells would be expected to contri­bute differentially to the differentiation of CD4 + T lym­phocytes, since fL-4 has been shown to promote a Th2 phenotype and IL-12 to promote a Thl phenotype. Since BM·MMC do transcrihe the Th2 cytokine IL-4 constitut­ively. we analyzed different mast cell populations for their ability to produce two other Th2 type cytokines, fL·13 and IL-IO. 2 Materials and methods 2.1 Cells and tissue culture BM-MMC were obtained hy culturing BM cells in IL-3- conditioned medium (IOO-30n ng/ml) derived from the supernatant of a 653 B cell line transfected with the mouse IL-3 eDNA sequence, as descrihed [16]. The cells were analyzed after 2.5-3 weeks of culture. BM-CTMC were ohtained by culturing BM cells with KL derived from the supernatant of CHO cells transfected with the mouse KL cDNA sequence. The KL-transfeeted CHO cell line was provided by Genetics Institute, Cambridge, MA. The BM· CTMC cclls were analyzed after 2.5-3 weeks of culture. Freshly isolated splenocytes and BM cells to be analyzed for IL-13 and fL-lO expression were obtained from out· bred NIH mice. Freshly isolated splenocytes were ohtained hy mincing the spleens in RPMI 1640 with 5 % FCS. resllspending and passaging through a nylon mesh to ohtain a single·cell sllspension. Activated thymocytes for the positive IL-13 control wcrc ohtained from the thymus of an outhred NIH mouse 2-+ h after intraperitoncal injection of Escherichia coli. 2.2 I'''ACS anulysis Biotinylated mAb used for FACS analysis were purchased from Pharmingell (San Diego. CA): fgG2c (Cat. no. Il042C), anti-c-Kit (Cat. no. OIH22D). and anti-CD3 (Cat. 77 Eur. J. Immunol. 1996.26: '+9-56 no. 01082D). Cell-surface staining was performed in the presence of mouse serum as described [16J. utilizing a FACScan flow cytometer (Becton Dickinson. Mountain View, CA). 2.3 Mast cell activation and IL·3 treatment Activation of BM-CTMC by IgE cross·linking was done as described by Tertian et al. [17J. using 20 ~lglml monoclonal anti-dinitrophenyl mouse IgE (Sigma, D-8-+06, St. LOllis, MO) for 30 min and subsequent addition of 100 ng/ml DNP·BSA for the time indicated in the figure legends. BM-derived mast celIs, splenocytes. and freshly isolated BM cells were stimulated with IL·3 by incubating the cells with IL-3 (l00-30n ng/ml) derived from the supernatant of 653 cells for the various times indicated in the figure legends. Cell concentrations for fL·] treatment as~ays were I x 10('-3 x ut cells/ml for BM-derived mast cells, and 5 x 10"_10 x lO" cells/ml for freshly isolated BM cells and splenocytes. 2.4 Rc\'crsc transcriptasc-rapid (ICR (RT·R)PCR analysis RNA from tissue samples was prepared hy using the CsCl1 guanidine method [IHI. Gene-specific transcripts were quantified hy the RT·RPCR protocol as descrihed [19.201. Annealing temperatures for eaeh oligonucleotide set arc listed helow. For all reactions, samples were denatured at 94°C for I s. annealed for I s and extended for to s at 72 0c. The gene-specific oligonucleotides used in this assay. the annealing temperatures lIsed during RT-RPCR. the size of the resultant products. sequence, and genc location arc listed in Tahlc t. 2.5 Intracellular staining Intracellular staining was performed by fixing the cells with 4 % paraformaldehyde for JO min, pcrmeabilizing the cells with the concentrations of saponin indicated in the figure legends, hlocking for 10 min with mouse serum, incuhating for 30-40 min with the antibodies indicatcd in the figure legends. and then an<llyzing with a FACSc<ln tlow cyto· meter. Cells were stained at densities of I x IOh-IO x 10" cells/ml with an antibody concentration of 5-10 ~lglml based on the method developed by Sander et al. [211. The mAh used were rat anti·mouse from Pharmin!!en (San Diego, CAl: anti·If:N-y (IgGI, clone XMGI.2), a~lti­TNF ·a (IgG I, clone MP6-XT22), anti·IL-1O (lgG I. clone JES5-2A5), and anti-IL·2 (lgG2a, clone JES6-IAI2). 2.6 Isolation or B220+ splel10cytes Separation of splenocytes into B220' and B22() popula­tions was performed using magnetic polystyrene heads (Dynal. Oslo, Norway; Dynabeads M-2RO: cat. no. 112.(5) covalently bound to streptavidin. Biotinylated rat anti· mouse antibody against B220 was bound to the Dynaheads according to standard instructions provided hy Dynal. The antibody against B220 was purchased from Pharmingen (cat. no. OI122A). Freshly isolated splenocytes wcre then 78 Eur. 1. Immunol. 1996.26: 49-56 IL-3 modulates the expression of fL-13 and IL-lO 51 Table 1. RT-RPCR oligonucleotidesdl , annealing temperature and expected product size Cytokine Annealing Expected Sequence of oligonucleotide primers temperature product size IL-1~ 60°C 209 bp 5' CAT GAG ACTTGC ACA GATCAG 3' 5' GGGTTG GAT GGT CTCTTC CAG 3' TGF-f3 55°C 150 bp 5' GATACC AACTATTGCTT3' 5' CCA AAT ATA GGG GCA GG 3' TNF-a 55°C 150 bp 5' CTC AGA TCA TCTTCT CA 3' 5' CAC CAC TAG TTG GTT GT 3 ' IL-13 60°C 240 bp 5' GGG TGA CTG CAG TCC TGG CT 3' 5' TGC AAT ATC CTCTGG GTC CT 3' ~-actin 60°C 135 bp 5' GTAACAATG CCATGTTCAAT3' 5' CTC CATCGTGGG CCG CTCTAG 3' fL-lO 60°C 246 bp ;' TCCTTAATG CAG GACTTT AAG GGTTACTTG 3' 5' GAC ACCTTG GTCTTG GAG CTT ATT AAAATC-3' IL-5 60°C 166 bp 5' ATG AGAAGG ATG CTTCTG CAC 3' 5' TGA GTA GGG ACA GGA AGC CTC 3' a) All gene sequcnces were obtained from GenBank submissions. The PCR products are designed to span one or more introns. incubated with the Dynabeads two times, each for 30 min at a concentration of 1 x 1011 Dynabeads/l x IOn spleno­cytes, and the B220+ cells extracted with a magnet. RNA was then extracted from the two resulting pools of cells using the CsCllguanidine method (18J. 3 Results 3.1 t'ACS analysis of 8M-derived mast cells We and others have demonstrated that cells possessing a mucosal mast cell phenotype can be derived from BM by long-term culture (>2 weeks) in the presence of IL-3 (22-26]. while a similar culture in KL produces mast cells possessing a connective tissue phenotype [16, 27-29]. For the experiments described in this report, it was important to assess the homogeneity of these two cell types. The c-kit tyrosine kinase receptor is known to be expressed on mast cells and their precursors. We tested the homogeneity of the BM-CTMC (differentiated in KL) and the BM-MMC (differentiated in fL-3) by staining the cells with antisera against c-kit or CD3 (Fig. 1). These analyses indicated that more than 95 % of the cells possess c-kit. while less than 5 % possess CD3. The cells analyzed in these experi- A. B. 90 90 Day 18 KL Derived Cells Day 181L·3 Derived Cells C-KIT C·KIT Figure I. Bone marrow-derived mast cells are c-kit' ICD3. (1-\) Bone marrow CTMC-like cells were cultured in c-kit ligand (KL) ror 18 days and then analyzed by flow cytometry using rat cOlllrol IgG2c antibody. hamster anti-mouse antibody agilinst CD3. or rat anti-mouse antibody against c-kit. (B) OM-MMC-like cells cul­tured in I L-3 for 18 days were analyzed by a FACScan flow cyto­meter with the nntibodies described above. ments were cultured for 18 days in their respective cyto­kines. Identical profiles were obtained with cells main­tained in culture for up to 45 days (data not shown), how­ever, freshly isolated BM was heterogeneous in the expres­sion of these two proteins (data not shown). 3.2 IL-13 expression by mouse mucosal mast cells IL-4 is an anti-inflammatory cytokine produced by Th2 cells, MMC and basophils. Since IL-l3 is similar to IL-4 in structure and function, we examined the expression of IL- 13 in BM-MMC and BM-CTMC (Fig. 2). We chose to ana­lyze these and other cells for cytokine transcripts using the RT-RPCR protocol developed in our lab [191. This proto­col allows for the analysis of many different gene products from a single mRNA/cDNA source. Additionally. by regu­lating the numbers of cycles used for product identifica­tion. we can ensure that the signal produced during ampli- Unact. Unact. Act. CTMC MMC Thymocytes ... ~~r('t).g !!!: ': d.d.tl .:.t- o •• M ~ til -u..u.. ..... U U - U. U. U ~(.!)Zt 'II ~~~~ 'II ·C)Z ~ til ::t :::::1-1-::::: c!:.. c!:.. :::::1-1- c!:.. • - • • -i. - J,. -• - ~r~ •• • • • • • Figure 2. Constitutive expression of I L-!3 transcripts by BM­MMC. Transcripts were analyzed by RT-RPCR from total RNA isolated from O:vt-CTMC-like cells cultured in KL for 20 days (Unnct. CTMC). BM-MMC-like cells cultured in IL-3 for 20 days (Unact. I'v1MC). and activated thymocytes (Act. Thymocytcs). For the cytokines IL-IB. TGF-11. TNF-n. and IL-13. the numher of RPCR cycles was 2R. nnd for B-aclin 16 cycles. 52 E, V. Marietta d aJ. unact. gOmin CTMC CTMC .. ~("l~ ::. 7 rt'l·5 u.. u.. - u ..:. u.. _ u ::t: ~ ~ ~ ~ C1 Z I t9 .......... ~ rb. 1. ~ ••;<I '~} - I• I· ,• I- • • 7hr. CTMC • • • Act. Thymocytes - • • Figure 3. Activated BM-CT\IC express IL-13 transcripts, RT­RPCR was used to analyze transcripts from the following RNA sampks: BM-CTMC-like cells that were cultured in medium alone for .:I. h (l!nact. CTMC). B\l-CTMC-like cells activated hy f.(:f1{1 cross-linkinl!. and maintained in culture for an additional l)() min (YO-min CTMC) or 7 h (7-h CTMC) and activated thvmo­cytes (Act. Thymocyks). All CT\lC-like cells used were cult'ured in KL for 20 days. RPCR cycle 1111mb..:rs w..:r..: 2X for th..: cytokin..:s TGF-fl, TNF-u. and IL-n. and Iii for II-actin. ficatioll is directly proportional to the quantity of tran­scripts sp('dfic for that product in any giv('n cDNA sampk [ 191. Transcript analysis of these two subsets of cells r('vealed cOllstitutive expression of IL-D transcripts in BM-MMC, but not in BM-CTMc' The quantity of I L-I.1 transcripts in the BM-MMC sample was similar to tht.: quantity of IL-13 transcripts expressed by activated thymocytes, indicating a physiologically relevant number of transcripts in BM­MMC. Ii-Actin transcripts WL're lIst.:d as normalization con­trols, while I l-I fi. TG F-fi, ami TNF-u were used to com­pare I L-13 transcript levcls with thost.: of other known mast ct.:11 cytokincs. 3.3 HM-CTJ\.IC expression of IL-B transcripts via FcdU activation As lkl1lonstrated ahove. B~I-CTMC did not constitLltiwly cxpress I L- U. Since cytokine e.xpression in mast cells can he induced via FcElH activation [30.311, we examined IL­D transcript t.:xpression in activated Bi\I-CTMC. This analysis showed a timt.:-dependt.:nt course (If transcript c.xpression with IL-D (Fig. J). That is. IL-I.1 transcripts appeared within 1.5 h after activation and disappeared 7 h post-activatillil. 3A BM-CT,,\IC exprl'SSiHI1 of 11..13 transcripts \'ia IL·3 treatment The previous experimt.:nt dt.:lllonstrakd that BM-CT\IC could he induced to express I L-n transcripts. J-[owevt.:r, BM-Mi\IC grown in I L-J possess I L-IJ transcripts in the abscnce of de~ranulation, su!.':!.':esting that another means of increasing I-L-IJ transcript;~xists~ To test whether I J.-J could directly inducl.! IL-IJ cxpression. BM-CTMC were treated with 11.-3 and analyzed f(lr I L-IJ transcripts 79 Eur. J. Immllnul. llNh. :!f): .. FI-56 A. B. D16 CTMC uc. uc. E ~~ ~ ~ >. 1-'" .s:: 1-'" u.., I-L; .c: U"" .c: ~~ N"'<"D' ri 0 0 - ... D. D+ « IL- 13 -... • • I L- 13 ;r actIn • • • I\-acti n Figure -I. IL-J tr..:atmt:nt increa;,..:s I L-13 tran;,cript k'vels in 13\1- CTMC. (A) RT-RPCR was ust:d to analyze transcripts pr..:scnt in tht: following RNA samples: Bivl-CTMC-likc cd Is cultured in IL-3 for 0, (l.5. 1.5. or ..j. h. Tho: B.M-CT:V1C-like cells had b..:t:n cui­tllrcu in KL for 16 days prior to fL-3 treatnwnt. (HI RT-RPCR was used to analyze transcripts from tht: following RNA samrles: BM-CT~fC-likc ct:lls cultured for 2l) uavs in KL alone (D2l) CTMC h53 Sup.). BM-CT7\lC-like cells·cllltur..:d for 29 days in KL with lL-J dcriveu from 653 supernatant (1)29 CTMC + 1i53 Sup.) for 4~ h. and activated thymocyt..:s (Act. Thyrn.). RPCR cyck numbers were 21-1 for [L-U and I h for B-actin. (Fig. 4A and B). IL-lJ transcripts were observable within the first hour after IL-3 addition and wert.: still present 4N h later. The same result was obtained when IL-3 from a variety of different sources was utilized (data not shmvnj. demonstrating that IL-J was the specific agcnt resulting in the incrt.:ase of IL-D transcripts. 3.5 IlM-CTMC expressiun of IL·to transcripts via IL·3 treatment I L-IO is a cytokine which shares a number of functions with that of IL-4 and IL-13. As sLlch, the cxpression of IL­lO might be expected to be correlated with that or IL-4 and I L-I3, especially by cells of the mucosal environment. Accordingly. we examined our mast cell poplliations for the expression or IL-lO. BM-MMC tkrived in fl-3 consti­tutively expressed I L-IO transcripts at low h.:vels (Fig. 5 A), as did thc 13M-CTMC. However, when the B~l­CTMC \vere shifted from a KL culture to one containing I L-3, a marked elevation of I L- J() transcripts was evidt.:nt. Compared to f)-actin, the levels of I L-lO transcripts in these cells wcrt.: very similar to thest.: of the CH 12. LX cell line [321, known to produce high kvds of IL-IO. The kinet­ics of thl' IL-3-dcpt.:ndent increase of IL-IO transcripts in BM-CTMC was determined. Tht.: .:xprcssion of I L- [() tran­scripts by these cells was rl'aliily observahk within ~ hand was maintained through 4K h. As a control, IL-5 did not show a similar induction. 3.6 11,-111 protein detected within HM·MMC and UM· CTMC Tht.: presence of IL-\.1 and IL-IO transcripts in a population of mast cells docs not guarantt.:e homogeneous expressillll of these gcnes (or their protein products) in all cells of the population. One method designed to analyze single ceils for thl'ir production of specific proteills. sllch as cytokines. is intracellular staining. Such an appro;lch was feasible for thl' analysis of IL-Ill but not IL-IJ. for which specific mAb applicable for intracellular staining have not bt.:en described. This procedure was first used to analyze B\I­M i\IC which constitutively express the IL-tO gene. As Ellr. J. Immllnol. I Y%. ]6: ..fl}-."ti ..., A. ... ~ + ~ u u u u N :::<:E: ::::::EE iu::<- : ui::<- : u::c "[L-IO • .~ ~-Actin -~ B. ..c: .'.."c.:: ...c:: ...c.;: .Q..c.(::) 0 "';v ~ v -.... [l-IO •• __ • lL-5 ••••• ~-Actin Figllre IL-J trL'atlllCl1t illcrL'a:-c:- IL-IO transcript 1L'\'\:1s in 13\'1- CTl\IC RNA isoiat..:d from Bl\I-deri\'ed mast cells was analvzed for gene-specific trallscripb. (A) B~I-~Ii\IC-like cells ll1aif1t~;ineLl in 11.-3 alone f(lr 16 davs (~Ii\lC), /H'vl-MMC-likc cclh ~i\cn an addition of KI. for ..fK h (MMC + KLJ, BM-CT\,IC-likc cells maintaincd in KL alone for 16 BM-CT\IC-like c<:lls givcll all addition (If Il-3 from h:'i3 supernatant for ..fK h ((,TMe 11.-.1), and CHI2.LX cells. RPCR l'ycle numhers Wl'rL' 30 for IL-IO and 16 for I~-al'tin. (B) Bivl-CTi'vIC-likc cells maintained in KL alonL' (II h). or t',iven 11.-.1 dcrivl'd from h:'iJ supernatant for 1.5 h,..f h. 2..f h m..f(~ h. Transl'riph analyzed w..:re 11.-10 (32 cydes). IL-5 (3i) cycles) and Iklctin (1(1 ..:y..:b). shown in Fig. 6, va rio liS concentrations of the permcahiliz­ing agent saponin were used to optimize staining. Plots A-I) demonstrate that increasing concentrations of sapo­nin increase the pl'fIlH:ahility of the cell and granule mem­branes. allowing fur a nOll-quantitative delllon:-.tratilln of the presence of the specific cytokinl's. The optimal rc:-.ponse. shown in plot C. was presumably due to the grealest penetration of antisera and least escape of intra­ccllular IL-W. Antisera against IFN-'( and Tf\iF-«( were lIsed as negative and positi\'e controls. respectivL'ly. These data indicate that the B:vt-l\fl\\C popUlation demonstrated homogeneolls expression of both I L-IO and TN F-u. A similar set of expL'riml'nts utilizing BM-CTlV\C treated with 11.-3 confirmed the level of stainin!!, of 1 L-lO antici­pated from the transcript analysis (Fig.~7). The staining shift for fL-)(I is greater tl1:111 two orders or magnitude 80 fL-3 mmlulat<;;s the expression ul'll-U and IL-!() A. B . 001', c, D. o 05'\" SaponHl Figure b. Il-IO is l'xpn:ssed hy B\I-dni\c:d rna~t cclls. InlraC\:lIu­lar staining \\-as u,>ed to as-.;"y I L-IO c:.xpre,,:-ion in individual n:II~. (A-D) BN\-f\.IMC-like cells Wl'n: cultured in 11.-.1 1'01'.1 week, and then analvzl'd hv flow CV!OI11L'trv lI:-in~ rat <lIlti-mOlIsc I~(; I anti­h( ldil's sp~cifi;.: 1';)1' IFN-';'. T"F-(-(' or 11.-10. The dilTerenr. "apunin CllTlCl'Iltration" utili/ed are indicated Ill.OIl) 0;', (A), lUll '~, (B). ().()5% (e). aTld 0.1');, (D)I· whcn comparing I1M-CTMC treated with 11.-.1 lor% h (B) with those 13!V\-CTMC cultured without IL<~ (A). A corre­spllnLiing neg.ative control, llsing antiserum against I L-2. was performed for both thc nq~ative and positive cell populations. Analysis of cells in plot 13 revealed two peaks of IL-IO staining suggesting heterogeneity in this pllpUIa­lion for membrane permeability. l'ytokine recognition. or both. However. hoth peaks dellwt]:-.trale a distinct pllsitivc shiftcornpared to uninLiuced 13M\IC (A) or the 11.-2 Ileg:l­live control in (B). 3.7 Inductinn of IL-13 and IL-IO transniption hy sph."lHqtcs and UM cclls hy IL-3 trcatmcnt As dl'scrihed above. I L-J was shown directly to increase [1.-13 and lL-to tran:-.cript levels in BM-CTMC. Activated T cells and dcgranulated mast cclls are the only sources or IL-IJ vet to he described, whereas lL-IO sources include activated Th2 cdls, thymocytcs. l11<lcroph:lges. and Ly I' B cells. To test whether l!.-J could increase tht: numher of A. ;l-r----------_, B. ~~---------------------, Fig/l/'(' 7. B:\I-CT\lC-like L'ell~ \\erL' cui· tured ill "L for .' week'> alld then WC:fe eithL'r cultured with 1 [,-3 for % h (IH or without 11.-3 (r\). 1'11;.: cell'> \\l'l'e thcll per· IlH:ahili/cd \\ith 11.1 '!!(\ ,apollill alld ana- 104 IVIed lw Il,lw ntol11L'trv U,ill!.! rat anti­I; HlIl'>e ,,'lltih(lliie,',pL'ufiL'for IL-~ (lI' 11.-11). E. V. ~farietta ct al. Splenocytes + IL-3 .c .c 111 .c 0 ..,. -- IL-13 --e TNF-u --e IL·10 --- ~-Actin ••• TGF-I) Bone Marrow + IL-3 .c .c 111 E 0 ..,. -- IL-13 .... _ TNF-a --. IL·10 _.. I)-Actin ••• TGF-I) Figurl' 8. IL·J im:reases 11.-1.3 and 11.-10 transcription in spleno­cytes and 13M eells. RFRPCR was used to analyze trallsnipts from the follO\villg R!'lA samples: (A) freshly isolated splenocyte,", or (B) 13M cells. both of which were trl'ated with 11,-3 deriv..:d from 6.:'3 supernatant for 0, I.:' or -I h. ({PCR eyd..: numners \vere 2)-; for IL·L'\. 21' for IL-[O. 2-1 forTGF·IL 2() forTNF-u, and 16 for I)·actin. IL-I3, IL-IO, or hoth transcripts in cell types other than mast cells, Wl' incuhatl'd cells freshly obtained from the spleen and BM ill IL-J for various lengths of time (thymo­cytes \Verl' also analyzl.'d hut possessed a high basal level of IL-I:\ transcription which was not influenced hy thl' addi· tion or exogenous IL-:1: data not shown). Butll the spleno­cyte (Fig. /{A) and bOl1e marrow cultures (Fig. XB) demonstrated significant increases in 11.-1.1 and IL-IO tran­script levels aft~r IL-J treatment. IL-U and IL-III tran­scripts appeared in the tr~ated splenic and 13M cultllr~s between 1.5 hand'" h, similar to that sel.'n for BM-Cil\fC. wherl.'as TGF-[) and TNF-(t \v~re unaffected. Although mast cdls an: also present within the spicens of mkc. they repres~nt less than I 'Y.. of the total cells [}}]. suggesting they alolle cannot he responsible for the increase in I L·13 and I L·IO transcript levels seen in these experiments. Splenic cells wer~ fractionated using mag­netic heads conjugated to either anti-B22IJ or anti-CD} antihodies. The resultant flmv·through cells were analyzed for 13.220' or CD.r staining cells. respectively. This analy­sis indicated the B220 selection procedure was more effi· cient f(lr the removal of cdls bearing this marker than the CD} proceuurl.' was for n.:mming CD}-bearing cells. Accordingly, splenic cells were divided into two pools (B220' allu B22(l') and transcript quantities analyzed, As shown in Fig. 9, the II.· 13 transcript levels were highest in the unhound (13.220 ) cells (Fig. 9, lane 4), whereas the Il- 10 transcript levels were highe