6
ImmunologyToday,VoL 10, No. I 1, 1989 r#,V'l#,14/S--n 19 Hercend, T., Meuer, S., Brennan, A. etaL(1983)J. Exp. Med. 158, 1547-1560 20 Thiele, D.L., Patel, S.S. and Lipsky, P.E.(1988) J. ImmunoL 140, 3253-3260 21 Hercend, T., Schmidt, T., Brennan, A. etaL (1984) Eur. J. Immunol. 14, 844-852 Z2 Uematsu, Y., Ryser, S., Dembic, Z. etaL (1988) Cell 52, 831-841 23 Patel, S.S., Wacholtz, M.C., Duby, A.D. etaL (1989)J. ImmunoL 143, 1108-1117 24 David, V., Bourge, J-F., Guglielmi, P. etaL (1987) J. ImmunoL 138, 2831-2836 25 Phillips, J.H., Weiss, A., Gemlo, B.T. etaL (1987)J. Exp. Med. 166, 1579-1584 26 Krensky, A.M., Sanchez-Madrid, F., Robbins, E. etaL (1983) J. ImmunoL 131,611-616 27 Schmidt, R.E., Bartley, G., Levine, H. etal. (1985) J. ImmunoL 135, 1020-1025 28 Meuer, S.C., Hussey, R.E., Fabbi, M. etaL (1984) Cell36, 897-906 29 Siliciano, R.F., Pratt, J.C., Schmidt, R.E. etal. (1985)Nature 317,428-430 30 Wacholtz, M.D., Patel, S.S. and Lipsky, P.E. (1989)FASEBJ. 3, A786 31 Brooks, C.G., Kuribayashi, K., Sale, G.E. etal. (1982) J. Immunol. 128, 2326-2335 32 Seaman, W.E., Talal, N., Herzenberg, L.A. etaL (1981) J. Immunol. 127, 982-986 33 Targan, S.R.and Newman, W. (1983) J. Immunol. 131, 1149-1153 34 Pawelec, G., Newman, W., Schwulera, U. et al. (1985) Cell. ImmunoL 92, 31-40 35 Gilbert, C.W., Zaroukian, MH. and Esselman,W.J. (1988) J. Immunol. 140, 2821-2828 36 Marvel, J. and Mayer, A. (1988) Eur. J. Immunol. 18, 825-828 37 Ledbetter, J.A., Tonks, N.K., Fischer, E.H. etal. (1988) Proc. Natl Acad. Sci. USA 85, 8628-8632 38 Streuli, M., Hall, L.R., Saga, Y. etal. (1987)J. Exp. Med. 166, 1548-1566 39 Clark, E.A. and Ledbetter, J.A. (1989) Immunol. Today 10, 225-228 40 Gruber, M.F., Bjorndahl, J.M, Nakamura, S. etal. (1989) J. ImmunoL 142, 4144-4152 41 Sanders, M.E., Makgoba, M.W. and Shaw, S. (1988) Immunol. Today 9, 195-199 42 Goto, M. and Zvaifler, N.J. (1985)J. ImmunoL 134, 1483-1486 43 Lanier, L.L., Le, A.M., Civin, C.I. etal. (1987)J. ImrnunoL 136, 4480-4486 Recent advances in the cellular and molecular Themastcell isnowconsidered to playa pivotalrolenotonly in biology of mast cells allergic reactions but also in a number of inflammatory dis- orders. After immunological activation via the IgE receptor, the mast cell releases a variety of cytokines, lipid-derived mediators, amines, proteases and proteoglycans - all of which can regulate adjacent cells and the metabolism of the extra- cellular matrix of connective tissues. While it had been known for some time that mast cells differ in a number of properties in varied tissue sites, it was not known why or how this hetero- geneity occurred. The development of in-vitro techniques to culture mast cells and the reconstitution of mast-cell-deficient mice are two major approaches that have facilitated analyses of how the tissue microenvironment regulates the phenotype of mast cells. In this review by Richard L. Stevens and K. Frank Austen, some of the recent findings on the molecular biology of mast cell secretory granule proteins and proteoglycans, and the interaction of mast cells with fibroblasts in the presence and absence of interleukin 3(IL-3) are highlighted. Heterogeneity of in-vivo and in-vitro differentiated mast cells Recent major advances in mast-cell biology have characterized many of the biological responses of acti- vated mast cells (Fig. 1). These cells constitute a heterogeneous population. It was first shown histo- chemically, and later morphologically, biochemically and functionally that the mast cells that are present in the Departmentof Medicine, Harvard MedicalSchool,and the Department of Rheumatologyand Immunology, Brigham and Women's Hospital, Boston, MA 02115, USA. Richard L. Stevensand K. Frank Austen gastrointestinal mucosa (mucosal mast cells or MMC) of helrninth-infected rats (Table 1) and mice are distinct from the population of mast cells that reside in the skin or the peritoneal cavity (connective tissue mast cells or CTMC) 1-1°. Ginsburg and co-workers 1~ derived a mixed population of mast cells, some of which synthesize heparin proteoglycans 12, by culturing dispersed lymph node cells from mice immunized with horse serum on a primary monolayer of mouse embryonic skin cells that morphologically resembled fibroblasts. In 1981, a num- ber of laboratories simultaneously discovered a more convenient and quantitative method for obtaining pure populations of murine bone-marrow-derived mast cells (BMMC) by culturing hematopoietic progenitor cells in conditioned medium derived from lectin-activated T cells. The cytokine in this T-cell conditioned medium that induces the preferential growth and differentiation of progenitor cells to mast cells is IL-3 (Ref. 13), but IL-4 (Ref. 14) can act in synergy with IL-3 to stimulate increased outgrowth of mast cells. Although these in- vitro differentiated mouse mast cells resemble mouse MMC in their T-cell factor dependence and in their histochemical properties of being alcian-blue-positive, safranin-negative cells, their secretory granules are more vacuolated than those in MMC, and thus their inter- relationship remains to be determined. Mouse BMMC © 1989, Elsevier SciencePublishersLtd, UK. 0167-4919/89/$03.50 381

Recent advances in the cellular and molecular biology of mast cells

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Page 1: Recent advances in the cellular and molecular biology of mast cells

Immunology Today, VoL 10, No. I 1, 1989

r#,V'l#,14/S--n 19 Hercend, T., Meuer, S., Brennan, A. etaL(1983)J. Exp. Med. 158, 1547-1560 20 Thiele, D.L., Patel, S.S. and Lipsky, P.E. (1988) J. ImmunoL 140, 3253-3260 21 Hercend, T., Schmidt, T., Brennan, A. etaL (1984) Eur. J. Immunol. 14, 844-852 Z2 Uematsu, Y., Ryser, S., Dembic, Z. etaL (1988) Cell 52, 831-841 23 Patel, S.S., Wacholtz, M.C., Duby, A.D. etaL (1989)J. ImmunoL 143, 1108-1117 24 David, V., Bourge, J-F., Guglielmi, P. etaL (1987) J. ImmunoL 138, 2831-2836 25 Phillips, J.H., Weiss, A., Gemlo, B.T. etaL (1987)J. Exp. Med. 166, 1579-1584 26 Krensky, A.M., Sanchez-Madrid, F., Robbins, E. etaL (1983) J. ImmunoL 131,611-616 27 Schmidt, R.E., Bartley, G., Levine, H. etal. (1985) J. ImmunoL 135, 1020-1025 28 Meuer, S.C., Hussey, R.E., Fabbi, M. etaL (1984) Cell36, 897-906 29 Siliciano, R.F., Pratt, J.C., Schmidt, R.E. etal. (1985)Nature 317,428-430 30 Wacholtz, M.D., Patel, S.S. and Lipsky, P.E. (1989)FASEBJ. 3, A786 31 Brooks, C.G., Kuribayashi, K., Sale, G.E. etal. (1982)

J. Immunol. 128, 2326-2335 32 Seaman, W.E., Talal, N., Herzenberg, L.A. etaL (1981) J. Immunol. 127, 982-986 33 Targan, S.R. and Newman, W. (1983) J. Immunol. 131, 1149-1153 34 Pawelec, G., Newman, W., Schwulera, U. et al. (1985) Cell. ImmunoL 92, 31-40 35 Gilbert, C.W., Zaroukian, MH. and Esselman, W.J. (1988) J. Immunol. 140, 2821-2828 36 Marvel, J. and Mayer, A. (1988) Eur. J. Immunol. 18, 825-828 37 Ledbetter, J.A., Tonks, N.K., Fischer, E.H. etal. (1988) Proc. Natl Acad. Sci. USA 85, 8628-8632 38 Streuli, M., Hall, L.R., Saga, Y. etal. (1987)J. Exp. Med. 166, 1548-1566 39 Clark, E.A. and Ledbetter, J.A. (1989) Immunol. Today 10, 225-228 40 Gruber, M.F., Bjorndahl, J.M, Nakamura, S. etal. (1989) J. ImmunoL 142, 4144-4152 41 Sanders, M.E., Makgoba, M.W. and Shaw, S. (1988) Immunol. Today 9, 195-199 42 Goto, M. and Zvaifler, N.J. (1985)J. ImmunoL 134, 1483-1486 43 Lanier, L.L., Le, A.M., Civin, C.I. etal. (1987)J. ImrnunoL 136, 4480-4486

Recent advances in the cellular and molecular Themastcell isnowconsidered to playa pivotalrolenotonly in biology of mast cells allergic reactions but also in a number of inflammatory dis- orders. After immunological activation via the IgE receptor, the mast cell releases a variety of cytokines, lipid-derived mediators, amines, proteases and proteoglycans - all of which can regulate adjacent cells and the metabolism of the extra- cellular matrix of connective tissues. While it had been known for some time that mast cells differ in a number of properties in varied tissue sites, it was not known why or how this hetero- geneity occurred. The development of in-vitro techniques to culture mast cells and the reconstitution of mast-cell-deficient mice are two major approaches that have facilitated analyses of how the tissue microenvironment regulates the phenotype of mast cells. In this review by Richard L. Stevens and K. Frank Austen, some of the recent findings on the molecular biology of mast cell secretory granule proteins and proteoglycans, and the interaction of mast cells with fibroblasts in the presence

and absence of interleukin 3(IL-3) are highlighted.

Heterogeneity of in-vivo and in-vitro differentiated mast cells Recent major advances in mast-cell biology have

characterized many of the biological responses of acti- vated mast cells (Fig. 1). These cells constitute a heterogeneous population. It was first shown histo- chemically, and later morphologically, biochemically and functionally that the mast cells that are present in the

Department of Medicine, Harvard Medical School, and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA 02115, USA.

Richard L. Stevens and K. Frank Austen

gastrointestinal mucosa (mucosal mast cells or MMC) of helrninth-infected rats (Table 1) and mice are distinct from the population of mast cells that reside in the skin or the peritoneal cavity (connective tissue mast cells or CTMC) 1-1°. Ginsburg and co-workers 1~ derived a mixed population of mast cells, some of which synthesize heparin proteoglycans 12, by culturing dispersed lymph node cells from mice immunized with horse serum on a primary monolayer of mouse embryonic skin cells that morphologically resembled fibroblasts. In 1981, a num- ber of laboratories simultaneously discovered a more convenient and quantitative method for obtaining pure populations of murine bone-marrow-derived mast cells (BMMC) by culturing hematopoietic progenitor cells in conditioned medium derived from lectin-activated T cells. The cytokine in this T-cell conditioned medium that induces the preferential growth and differentiation of progenitor cells to mast cells is IL-3 (Ref. 13), but IL-4 (Ref. 14) can act in synergy with IL-3 to stimulate increased outgrowth of mast cells. Although these in- vitro differentiated mouse mast cells resemble mouse MMC in their T-cell factor dependence and in their histochemical properties of being alcian-blue-positive, safranin-negative cells, their secretory granules are more vacuolated than those in MMC, and thus their inter- relationship remains to be determined. Mouse BMMC

© 1989, Elsevier Science Publishers Ltd, UK. 0167-4919/89/$03.50

381

Page 2: Recent advances in the cellular and molecular biology of mast cells

-reviews Immunology Today, Vol, 10, No. I l, 1989

LIPID MEDIATORS

PGD2

/ "~ ~ - - ~ ' ~ Q I ' }%~im-,-~ SECRETORY GRANULE " . . .... . i ":ii! \ PREFORMED MEDIATO_RS

/ : ~ ::~ • Histamine !~t • Proteoglycons !ii:~= • Serine proteases

• Carboxypeptidose A \::,: ! ~:~7:. ~:::: i: 'e ::::" i : : ~ CYTOKINES

\:.! :/Qi::. :: l:::Oi L-3

• IL -6 Acl/vofed Mos! Cell • GM-CSF

LEUKOCYTE RESPONSES • Adherence • Chemotoxis • IgE production • Most cell proliferation • Eosinophil activation

FIBROBLAST RESPONSES

• Proliferation • Vocuolotion • Globopentaosylceramide production • Collagen production

SUBSTRATE RESPONSES

• Degradation of proteins (e.g. fibronectin, Type ]3" collagen,

and lipoprofein$) • Activation of coagulation cascade

MICROVASCULARRESPONSES

• Augmented venular permeability • Leukocyte adherence • Constriction • Dilatation

Fig. 1. Biological responses of activated mast cells. When the mast cell lgE receptor is cross-linked with antigen, the mast cell becomes activated and releases preformed secretory granule mediators and newly synthesized cytokines and plasma-membrane-derived lipid mediators. Some of the biological responses of the activated mast cell are illustrated to demonstrate the diverse functions of this novel cell.

3 8 2

are phenotypically very different from those CTMC that are isolated from the peritoneal cavity. BMMC have been used to identify and characterize mouse mast cell proteo- glycans ls,16, serine proteases 16,17, carboxypeptidase A (Ref. 18), glycolipids 19, plasma membrane proteins 2° and arachidonic acid metabolites 21,22. They have also been used to study intracellular trafficking of molecules 23, post-translational modification of mast cell proteins 2° and proteoglycans 23, macromolecular complex forma- tion in the secretory granules 16,18, different stages of mast cell differentiation 24-26, and the activation- secretion responses of mast cells 21,22,2s,27.

Haig and co-workers 28 and then Broide and co- workers 29 obtained a population of rat mast cells that appeared to be histochemically, morphologically, bio- chemically and functionally almost identical to rat MMC by culturing hematopoietic progenitor cells in con- ditioned medium derived from lectin-activated T cells (Table 1). These culture-derived rat BMMC were similar to T-cell-dependent rat MMC in that they possessed electron-dense granules that stained alcian-blue-positive, safranin-negative. The rat BMMC were also found to be similar to rat MMC in that both populations of cells con- tained rat mast cell protease-II (RMCP-II) and chondroitin sulfate diB proteoglycan in their secretory granules, and generated both prostaglandin D2 and leukotriene C4 when they were immunologically activated 6-9,29.

Metachromatic, histamine-containing IgE-receptor- bearing cells have also been obtained when human cord blood progenitor cells 3° or bone marrow cells 31 were cultured in the presence of recombinant human IL-3 or conditioned medium from lectin-activated T cells. How- ever, as assessed by their ultrastructure and plasma membrane phenotype, these cells resemble immature basophils more closely than mast cells.

The deve lopmenta l re lat ionship of mast cells Because of the substantial differences in the pheno-

types of rat (Table 1) and mouse CTMC relative to MMC, it was assumed that MMC and CTMC were not related in their differentiation. However, proteoglycan biosynthetic experiments with and without 13-D-xyloside 23 suggest that the two populations of mast cells are more similar to one another in their differentiation than was previously appreciated. Rat basophilic leukemia-1 cells 32 and rat BMMC 29 synthesize [3SS]proteoglycans that bear pre- dominately chondroitin sulfate diB glycosaminoglycans; rat MMC synthesize [3SS]proteoglycans that have chon- droitin diB glycosaminoglycans and lesser amounts of chondroitin sulfate E glycosaminoglycans6; and mouse BMMC synthesize [3SS]proteoglycans that have almost exclusively chondroitin sulfate E (Refs 15, 16). Although rat peritoneal CTMC synthesize [3sS]proteoglycans that bear only heparin glycosaminoglycans, when these CTMC are exposed to the exogenous glycosaminoglycan acceptor, p-nitro-phenyl-13-D-xyloside, they continue to synthesize [3sS]heparin onto the peptide core, but syn- thesize predominately [3SS]chondroitin sulfate E onto the acceptor 23. This in-v i t ro evidence for a dual biosynthetic capacity for distinct glycosaminoglycans in rat CTMC suggests that the phenotype of mast cells may not be fixed, but instead may be dependent on external factors such as the tissue microenvironment and/or their stage of differentiation.

In-vivo reconstitution experiments by Kitamura and co-workers using mast-cell-deficient W/W v mice, showed that parenteral injection of mouse BMMC could give rise to intestinal MMC and to skin (and peritoneal cavity) CTMC (Ref. 33), and that cultured peritoneal CTMC could give rise to local cells that histochemically re- sembled MMC when injected into the stomach of the

Page 3: Recent advances in the cellular and molecular biology of mast cells

Immunology Today, VoL 10, No. 11, 1989

mutant mouse 34. They concluded that the tissue micro- environment regulated the histochemical phenotype of mast cells, and that it was possible to alter the phenotype of mouse mast cells in vivo. Studies on SI/Sl d mice indicated that this latter type of mast-cell-deficient animal lacked skin CTMC because of a defect in their tissue microenvironment rather than because of an abnormality in their progenitor cells 35. Although the view that a highly differentiated cell such as the CTMC can dramatically alter its phenotype may seem novel, it is well known that connective tissue producing cells can alter their phenotype under different in-vitro and in-vivo conditions. For example, when a type II collagen- producing chondrocyte is placed on a plastic culture dish and allowed to flatten, it quickly converts to a type I collagen-producing fibroblast. However, if the fibroblast- like cell is then cultured in agarose gels it reverts back to a type II collagen-producing chondrocyte 36.

Although the above studies suggest that mast cell heterogeneity in rodents is probably not a consequence of an irreversible divergent differentiation of stem cells occurring in the bone marrow, knowledge of how heterogeneity of mast cells develops in humans is less clear. Functional37, 38 and chemical 39,4° differences have now also been noted in varied populations of human mast cells. Two populations of human mast cells have been distinguished based on the presence or absence of a chymotryptic protease (designated as chymase). Chymase-negative mast cells are decreased in the gastro- intestinal mucosa of patients with defective T lympho- cytes, but the number of chymase-positive mast cells in the submucosa is not 41. Although these findings led to the proposal that early divergent pathways exist for the development of these two populations of human mast cells, they do not exclude the possibility of transdifferen- tiation of the two subclasses of human mast cells.

Fibroblast influence on mast cell phenotypic properties The development of in-vitro technology 13 to obtain a

population of rodent mast cells that could be used to reconstitute all of the mast cells in mast-cell-deficient W/W v mice 33,34 encouraged the search for an in-vitro method for obtaining CTMC-like mouse mast cells. It had been simultaneously recognized that the viability of purified rat 42 and mouse peritoneal CTMC could be maintained for at least 30 days when they were co- cultured ex vivo with a monolayer of Swiss albino mouse 3T3 fibroblasts in the absence of growth factors-other than those in fetal calf serum. If these CTMC were not exposed to the fibroblasts, all of the mast cells died within 48 hours, even if mouse IL-3 was present in the culture medium. When they were co-cultured with fibroblasts, the purified rat peritoneal CTMC remained alcian-blue-positive, safranin-positive and continued to synthesize [3sS]heparin proteoglycans. The co-cultured CTMC released prostaglandin D2 and histamine when activated via their IgE receptor, and they possessed se- cretory granules that were as electron dense as the freshly- isolated cells. The morphological studies also showed that the two cell types approached to within 20 nm of each other and seemed to interact through their plasma membrane microplicae.

Because CTMC are fibroblast-dependent mast cells whereas MMC and BMMC are T-cell-dependent mast cells, non-cloned 24 and cloned mouse BMMC were

I Table 1. Characteristics of different populations of rat mast cells

Property CTMC MMC BMMC RBL-1 cell

Histochemistry safranin + T-cell factor dependence No Secretory granule electron +++++

density Major proteoglycan heparin Serine protease RMCP-I Histamine (1~g/106 cells) 10-30 IgE receptor Yes Arachidonic acid PG D2

metabolite Activation by 48/80 Yes

safranin- safranin- safranin- Yes Yes No (tumor cell)

+++++ ++ +

ChS-diB ChS-diB ChS-diB RMCP-II RMCP-II RMCP-II 0.1-1 1-2 -0.1 Yes Yes Yes

LTC4>PGD2 LTC4=PGD2 LTC4>PGD2

No No No

co-cultured with mouse 3T3 fibroblasts in the presence or absence of IL-3. In all instances, the alcian-blue- positive, safranin-negative mouse BMMC changed their histochemical phenotype after approximately 10 days of co-culture to become hybrid-staining cells in which some of the granules of an individual cell were alcian-blue- positive, safranin-positive while other granules remained alcian-blue-positive, safranin-negative. In many of the 28 day co-cultures, all of the granules of the mast cells became alcian-blue-positive, safranin-positive. This histo- chemical change was associated with a dramatic alter- ation in the biosynthesis of secretory granule proteo- glycans 24. While the starting population of mouse BMMC synthesized [35S]chondroitin sulfate E proteo- glycans and [3sS]heparin proteoglycans at an approxi- mate ratio of 50:1, the fibroblast co-cultured BMMC synthesized these proteoglycans at an approximate ratio of 1:3. It has been shown that a single gene encodes the peptide core of probably all secretory granule proteo- glycans present in effector cells that originate from the bone marrow 43. Thus, depending on the microenviron- ment and/or the stage of differentiation of the mouse mast cell, the proteoglycan peptide core is post- translationally glycosylated with either chondroitin sul- fate E glycosaminoglycans or heparin glycosamino- glycans.

After four weeks of co-culture, the electron density of the granules had noticeably increased and the mast cells contained approximately 50-fold more histamine and approximately 100-fold more carboxypeptidase A activity than the starting BMMC (Ref. 18). When activated by calcium ionophore or by antigen perturbation of their IgE receptors, co-cultured mouse mast cells that were adher- ent to the layer of fibroblasts exocytosed a higher percentage of their histamine and generated more arachidonic acid metabolites than the non-co-cultured mouse BMMC (Ref. 25). Like normal mouse CTMC, the co-cultured mast cells also expressed the Forssman glyco- lipid epitope, globopentaosylceramide, on their plasma membrane 26. If mouse BMMC were co-cultured with fibroblasts in the absence of IL-3, they changed their histochemical phenotype and increased their synthesis of [3sS]heparin proteoglycans relative to [3SS]chondroitin sulfate E proteoglycans, but did not increase their hista- mine content and did not multiply 24. These in-vitro studies clearly indicated that 3T3 fibroblasts had a dramatic time-dependent influence on the phenotypic 383

Page 4: Recent advances in the cellular and molecular biology of mast cells

e Immunology Today, Vol. 10, No. 11, 1989

3114

properties of mouse mast cells. Recently, a factor has been identified in 3T3 fibroblast

conditioned medium that stimulates the proliferation and differentiation of mouse progenitor cells into cells that histochemically resemble mouse CTMC (Ref. 44). Fibroblasts produce a diverse array of cytokines, includ- ing granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, IL-1 and IL-6 (Refs 45-48). However, because IL-1, IL-2, IL-4, GM-CSF, G-CSF, M-CSF, interferon (IFN), nerve growth factor (NGF) and epithelial growth factor (EGF) could not induce pro- genitor cells to undergo this phenotypic change 44, the fibroblast-derived factor that regulates the differen- tiation of hematopoietic stem cells into CTMC remains to be determined.

Immortalization of rat and mouse mast cells Although the fibroblast co-culture system repre-

sented a significant advance for studies on the regulation of mouse mast cell phenotypes, the period of 3-4 weeks needed to induce some or most of the mouse BMMC to become similar to CTMC, the different rate at which individual BMMC changed their phenotype, and the cumbersome task of separating the two cell types from one another, limited the use of these in-vitro derived mast cells for certain types of experiments. Chemical mutagenesis and viral transfection represented two alternative approaches to obtain large numbers of im- mortalized rodent mast cell lines. A leukemia that was composed of basophilic cells was generated in rats using the carcinogen 13-chlorethylamine 49. The cell line (RBL-1) that was established from this leukemia has been used to characterize the rat IgE receptor s°,sl, the steps involved in activation-secretion responses 52, and the arachidonic acid metabolites that are generated during cellular activation 53. On the basis of their alcian-blue-positive safranin-negative histochemistry, the ultrastructure of their secretory granules, and the presence of RMCP-II and chondroitin sulfate diB in their secretory granules, these chemically-immortalized cells closely resemble rat MMC (Refs 32,54; Table 1).

Ihle and co-workers obtained highly-proliferative im- mortalized mouse mast cell lines that were histochemi- cally and biochemically similar to IL-3-dependent mouse BMMC by transfecting hematopoietic progenitor cells with either Abelson murine leukemia virus 55 or Harvey sarcoma virus. Recently, fifteen separate mouse mast cell lines were immortalized with the ras oncogene by co- culturing mouse splenocytes in the absence of T-cell- derived factors with fibroblasts that were producing Kirsten sarcoma virus pseudotyped with the amphotropic leukemia (helper) virus 4070 (Ref. 56). These Kirsten sarcoma virus-immortalized mast cell lines (KiSV-MC) represented a range of histochemical and biochemical phenotypes. A correlation was observed between the percentage of safranin-positive granules in the cell, and the carboxypeptidase A content, histamine content, and biosynthesis of [3sS]heparin proteoglycans relative to [3SS]chondroitin sulfate E proteoglycans. All of the KiSV-MC cell lines had more differentiated granules than mouse BMMC. Several of the cell lines were found to be very similar to mouse peritoneal cavity CTMC in terms of their histochemistry, ultrastructure, histamine content, carboxypeptidase A content, and biosynthesis of [3sS]heparin proteoglycans.

Molecular biology of mast cell proteins and proteoglycans The development of these transformed (RBL-1 cell and

KiSV-MC) and non-transformed (BMMC) rat and mouse mast cell lines has allowed investigators to identify and characterize at the molecular level those proteins that are specifically expressed in mast cells of these rodents. RBL- 1 cell-derived cDNA libraries have been used by two groups to isolate cDNAs that encode the OL subunit of the high-affinity rat IgE receptor s°,sl. One of these RBL-1 cell cDNA libraries has also been probed with synthetic [32p]oligonucleotides that were constructed based on the amino acid sequence of RMCP-II (Ref. 7) to isolate a cDNA that encodes this rat MMC/RBL-1 cell secretory granule serine protease 57. Analysis of the deduced amino acid sequence of the cDNA showed that the translated mast cell prepro-enzyme consists of 247 amino acids with a typical 18 amino acid signal peptide. The pro-enzyme is predicted to differ from the stored se- cretory granule enzyme in that it has two additional glutamic acid residues at its amino terminus and three additional amino acids (Thr-Ser-Ser) at its carboxy terminus. Although the significance of the proteolytic processing of the carboxy terminus of RMCP-II is un- known, elastase undergoes a similar type of carboxy- terminal processing in the secretory granule of the human neutrophi158. The proteolytic cleavage at the amino terminus is a common occurrence in the post- translational modification of serine proteases, and results in activation of the inactive zymogen. A rat liver genomic library was probed with the RMCP-II cDNA to isolate and characterize this mast cell gene 59. Because a 5' flanking region of the RMCP-II gene was found to be homologous to a cis-acting regulatory element in a pancreatic serine protease gene, different populations of cells were transi- ently transfected with constructs that contained the putative regulatory element of the RMCP-II gene ligated to the human growth hormone gene. Not only did these experiments confirm that this 5' flanking region of the RMCP-II gene contained an element that enhanced transcription of this gene in mast cells, but the studies also indicated that this element did not enhance tran- scription in Rat-2 cells (rat fibroblasts), L cells (mouse fibroblasts), and M-12 cells (mouse B lymphoma cells).

The amino-terminal amino acid sequences of a serine protease 6° and a carboxypeptidase 61 that are both present in the secretory granules of mouse KiSV-MC have been determined. A cDNA library has been pre- pared from KiSV-MC and used to isolate distinct cDNA that encode these novel enzymes. The KiSV-MC-derived serine protease cDNA encodes a 28 kDa basically-charged enzyme whose substrate preference on the basis of the binding region is predicted to be chymotryptic rather than tryptic. Recently, this serine protease has been shown to be expressed selectively by those mast cells (MMC) that are present in the proximal small intestines of mice infected with Nippostrongylus brasiliensis. The KiSV-MC-derived carboxypeptidase cDNA encodes a basically-charged 36 kDa protein whose substrate pref- erence is predicted to be more similar to pancreatic car- boxypeptidase A than carboxypeptidase B. As assessed by protein sequencing of the isolated protein and by RNA blot analysis of different populations of cells, this novel carboxypeptidase has been shown to be a prominent secretory granule enzyme of mast cells of the CTMC subclass.

Page 5: Recent advances in the cellular and molecular biology of mast cells

Immunology Today, VoL 10, No. 11, 1989

.re¢ietus- Mouse BMMC have more mRNA per cell than rat

peritoneal CTMC, mouse peritoneal CTMC, or human lung mast cells, and therefore they are an excellent source of a pure population of mast cells for identifying those genes that are specifically expressed in non- transformed mouse mast cells. A cDNA library prepared from BMMC-derived mRNA has been used to isolate a cDNA, which was then used to isolate the gene encoding the peptide core of mouse secretory granule proteoglycans 62. Based on the deduced amino acid sequence of this gene, the mouse BMMC proteoglycan peptide core is translated as a 16.7 kDa protein which contains a twenty-one amino acid glycosaminoglycan attachment region consisting of alternating serine and glycine. When the predicted amino acid sequence of the mouse BMMC proteoglycan peptide core was compared with the predicted amino acid sequences of the homolo- gous rat 63 and human 64 molecules, the amino terminus was found to be highly conserved, indicating that this region of the peptide core may be important for the structure, function and/or metabolism of this family of proteoglycans. Rat-1 fibroblasts have been co- transfected with the selectable marker pSV2 neo and a clone containing this mouse gene to obtain fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. Since a 1.0 kb secretory granule proteoglycan peptide core mRNA transcript was detected in these transfected fibroblasts, it was concluded that the isolated mouse genomic clone contained the entire mouse secretory granule proteo- glycan peptide core gene, including some of the regulat- ory elements in its promoter region. Highly conserved nucleotide sequences have been identified within the 5' flanking region of the human and mouse secretory granule proteoglycan genes, which suggests that this region contains conserved cis-acting regulatory elements that are important for expression of this gene in hemato- poietic cells.

Effect of IL-3-dependent mouse BMMC on fibroblasts Evidence has been obtained that during co-culture in

the presence of IL-3, not only do the mast cells change their phenotype towards CTMC but the fibroblasts also undergo a number of changes. Morphological studies revealed that the fibroblasts became vacuolated and less adherent to plastic culture dishes when they were co- cultured with mouse BMMC for 14 days in the presence of 50% WEHI-3 conditioned medium as a source of IL-3 (Ref. 25). Since in-vivo differentiated mouse peritoneal CTMC contain globopentaosylceramide on their surface, it is not surprising that the co-cultured BMMC become globopentaosylceramide-positive. Biosynthetic exper- iments, however, showed that only the fibroblasts were synthesizing this glycolipid 26. Thus, it was concluded that, in the presence of IL-3, mouse BMMC induced the fibroblasts to increase their synthesis of globopentaosyl- ceramide with subsequent transfer to the plasma mem- brane of the mast cells. These studies on the membrane expression of globopentaosylceramide provided the first definitive evidence that a complex bilateral interaction was occurring between the two co-cultured cell types in which both were altered.

It was subsequently shown that the fibroblasts lost their contact inhibition and increased their growth rate when they were exposed to BMMC that were proliferat-

ing in response to IL-3 (Ref. 65). After four weeks of co-culture, up to eleven layers of fibroblasts were de- tected in some of the culture dishes. Because of this increase in the number of fibroblasts, there was an increased transcription of the type I collagen gene and an increased rate of biosynthesis of total collagen per culture. Recent studies have revealed that mouse BMMC and certain mouse mast cell lines produce GM-CSF, IL-3, IL-4, IL-5 and IL-6 in response to calcium ionophore or cross-linkage of their IgE-receptor 66,67. Thus, it is poss- ible that one of these mast-cell-derived cytokines may influence the fibroblast during co-culture. Because the number of mast cells increases in many different fibrotic conditions 68, it has been suggested that mast cells play a prominent role in fibrosis. The BMMC-fibroblast co- culture studies suggest that a particular subset of mast cells that are in an IL-3-activated state may be respon- sible for inducing fibroblasts to multiply and for the tissue to accumulate extracellular matrix. This hypothesis might explain why extensive fibrosis occurs in the bone marrow, liver and spleen, but not in the skin, of masto- cytoma patients 69. More importantly, these mast cell- fibroblast co-culture studies point to a positive role for certain populations of mast cells in repair of damaged connective tissue.

References 1 Enerb~ck, L. (1966)Acta Pathol. Microbiol. Immunol. Scand. 66, 303-312 2 Befus, A.D., Pearce, F.L., Gauldie, J., Horsewood, P. and Bienenstock, J. (1982) J. Immunol. 128, 2475-2480 3 Everitt, M.T. and Neurath, H. (1980)FEBS Lett. 110, 292-296 4 Lagunoff, D. and Pritzl, P. (1976)Arch. Biochem. Biophys. 173, 554~-563 5 Yurt, R.W., Leid, R.W., Jr, Austen, K.F. and Silbert, J.E. (1977) J. Biol. Chem. 252, 518-521 6 Stevens, R.L., Lee, T.D.G., Seldin, D.C. etaL (1986) J. Immunol. 137,291-295 7 Woodbury, R.G., Katunuma, N., Kobayashi, K., Titani, K. and Neurath, H. (1978) Biochemistry 17, 811-819 8 Trong, H.L, Parmelee, D.C., Walsh, K.A., Neurath, H. and Woodbury, R.G. (1987)Biochemistry 26, 6988-6994 9 Heavey, D.J., Ernst, P.B., Stevens, R.L. etal. (1988) J. Immunol. 140, 1953-1957 10 Lewis, R.A., Sorer, N.A., Diamond, P.T. etal. (1982) J. Immunol. 129, 1627-1631 11 Ginsburg, H. and Sachs, L. (1963) J. NatlCancerlnst. 31, 1-40 12 Bland, C.E., Ginsburg, H., Silbert, J.E. and Metcalfe, D.D. (1982) J. BioL Chem. 257, 8661-8666 13 Ihle, J.N., Keller, J., Oroszlan, S. etal. (1983) J. ImmunoL 131,282-287 14 Mosmann, T.R., Bond, M.W., Coffman, R.L., Ohara, J. and Paul, W.E. (1986) Proc. NatlAcad. Sci. USA 83, 5654-5658 15 Razin, E., Stevens, R.L., Akiyama, F., Schmid, K. and Austen, K.F. (1982)J. Biol. Chem. 257, 7229-7236 16 Serafin, W.E., Katz, H.R., Austen, K.F. and Stevens, R.L. (1986) J. Biol. Chem. 261, 15017-15021 17 DuBuske, L., Austen, K.F., Czop, J. and Stevens, R.L. (1984) J. Immunol. 133, 1535-1541 18 Serafin, W.E., Dayton, E.T., Gravallese, P.M., Austen, K.F. and Stevens, Ri. (1987)J. Immunol. 139, 3771-3776 19 Katz, H.R., Schwarting, G.A., LeBlanc. P.A., Austen, K.F. and Stevens, R.L. (1985) J. Immunol. 134, 2617-2623 20 Katz, H.R. and Austen, K.F. (1987)J. Immunol. 138, 1196-1200 21 Razin, E., Mencia-Huerta, J-M., Stevens, R.L. etal. (1983) J. Exp. Med. 157, 189-201 385

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386

Human Immunogenetics

edited by Stephen Litwin, Dekker, 1989. $150.00 (USA and Canada), $180.00 (all other countries) (856 pages) ISBN

0 8247 7899 5

This book contains a great deal of useful information. It suffers, how- ever, in taking too broad a view of the term ' immunogenetics'. The thirty-two chapters try to cover too diverse a range of topics, resulting in a volume that reads as a collection of disconnected chapters without any unifying theme and, therefore, depth. ' lmmunogenetics' is here considered to encompass: (1) any use of specific antibody probes to

study products of the human genome; (2) any genes controlling immune responses; (3) genes encod- ing tumour-associated antigens (as detected by antibodies) and (4) use of antibodies to study polymorphic systems. If this was not enough, there are also contributions on proto-oncogenes and breast cancer, human T-lymphotropic viruses and the mouse T-t complex.

Sadly, for a book on this theme published in 1989, there is very little about genes encoding the T-cell re- ceptors, where there has been an explosion of knowledge. Further- more as the detailed structure of major histocompatibility complex (MHC) class I and class II molecules

has emerged since the chapters were written, there is an 'aged' look to the discussions on the genes encoding T-cell responses and the basis of MHC association with certain dis- eases. Perhaps the most significant omission is any assessment of the clinical relevance of MHC matching in the areas of human organ and marrow transplantation and after all, there is a wealth of information in that area.

The book is organized into five parts. The first part covers "Approaches and tools of im- munogenetics". Some of the contri- butions are useful and well thought out. Even within this methodologi- cal/defining-the-rules-section, there