Anti-RMA, a Murine Monoclonal Antibody, Activates Rat Macrophages: II. Induction of DNA Synthesis and Formation of Multinucleated Giant Cells David Lazarus, Moshe Yamin, Karin McCarthy, Eveline E. Schneeberger, and Richard Kradin Immunopathology and Pulmonary Units of the Departments of Pathology and Medicine, Massachusetts General Hospital, Boston, Massachusetts

Anti-RMA is a murine anti-rat monoclonal antibody that binds to a 120-kD surface membrane antigen expressed primarily by alveolar macrophages. Saline-lavaged alveolar macrophages (AM) formed clusters after incubation with anti-RMA. Anti-RMA produced multinucleated giant cells (MGC) in rv15 % of adherent AM, and the F (ab'), fragment of anti-RMA yielded MGC in rv9% of AM. The Fab fragment of anti-RMA did not promote MGC formation, nor did the murine anti-rat monoclonal antibodies OX41 and W3/25 (anti-CD4). Although anti-RMA produced a tenfold increase in PH]thymidine incorporation by AM, it yielded a minimal increase in the number of AM. Autoradiography of AM stimulated with anti-RMA showed heterogeneous labeling of nuclei in MGC, suggesting that 3H-Iabeled AM may fuse with AM that are not actively synthesizing DNA. These findings suggest that binding of anti-RMA to AM may activate DNA synthesis, and promote clustering and fusion of AM, leading to MGC formation.

Macrophages are multifunctional cells that participate in the inflammatory response (1-4). After exposure to certain bacteria, viruses, and foreign bodies, some macrophages form multinucleated giant cells (MGC) (5-10). MGC are a feature of the granulomatous inflammation which characterizes a variety of noninfectious human pulmonary diseases, including sarcoidosis and hypersensitivity pneumonitis (11, 12). MGC may be produced by incubating mononuclear phagocytes in conditioned supernatants of cultured lymphocytes (13-16) and in recombinant interferon-gamma (IFN-')') (17) and interleukin-4 (IL-4) (18). Substances other than lymphokines, including the steroid hormone 1,25 dihydroxyvitamin D (19), also augment MGC formation by macrophages. MGC have been demonstrated to form as a result of macrophage fusion (11). Although fusion of macrophages may be the primary mode of MGC formation, nuclear reproduction in the absence of cell division may also play a role (20). However, the mechanisms leading to MGC formation are currently uncertain. We have developed a murine monoclonal antibody Key Words: alveolar macrophages, multinucleated giant cells, DNA synthesis, granuloma (Received in original form October 6, 1989 and in final form March 12, 1990) Address correspondence to: Richard Kradin, M.D., Immunopathology Unit, Cox 5, Massachusetts General Hospital, Boston, MA 02114. Abbreviations: alveolar macrophages, AM; fetal calf serum, FCS; interferon-gamma, IFN--y; inter1eukin-4, IL-4; lipopolysaccharide, LPS; monoclonal antibody, mAb; multinucleated giant cells, MGC; phorbo1 myristate acetate, PMA. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 103-111, 1990

(mAb), anti-RMA (IgG1K), that binds to a surface membrane antigen expressed primarily on rat alveolar macrophages (AM) (21). In this article, we report that anti-RMA promotes clustering, DNA synthesis, and MGC formation by AM.

Materials and Methods Animals Pathogen-free, 6- to 10-wk-old female Lewis rats (150 to 250 g) were obtained from Charles River Laboratories (Kingston, MA). Animals were housed in the restrictedaccess research animal care facility at Massachusetts General Hospital and were permitted access to food and water ad libitum. Isolation of Bronchoalveolar Macrophages Animals were anesthetized with i. p. injections of sodium pentobarbital, 0.05 g/kg. A short piece of 0.034-inchdiameter polyethylene tubing was inserted into the trachea and secured with a tight suture. Following bilateral diaphragmatic incisions, sterile PBS, pH 7.3, without calcium or magnesium, was injected in 5-ml aliquots, allowed to dwell for 15 s with chest massage, and subsequently withdrawn. A total of 25 to 30 ml were infused into each animal, with a return of 80 to 90% of the instilled volume, consistently yielding 2 to 4 x 1Q6 AM/animal. Lavage fluid was pooled in 50-ml plastic centrifuge tubes (Falcon; BectonDickinson, Lincoln Park, NJ), centrifuged at room temperature at 400 x g, and the cell pellet was washed 3 times in PBS. AM constituted more than 90 % of the cells as judged by their morphology and their ingestion oflatex beads. After the final wash, the cell pellet was suspended in RPMI-1640

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with 5 % heat-inactivated FCS and 1% penicillin and streptomycin solution (all from GIBCO, Grand Island, NY). Preparation of Fab and F(ab')2 Fragments Anti-RMA was produced as described elsewhere (21), and Ftab'), fragments of anti-RMA were prepared by enzymatic digestion with papain (22). The Flab'), fragments were isolated on a DEAE-cellulose column (DE-52; Whatman Labatory Products, Clifton, NJ) equilibrated with 0.005 M Tris-HCI buffer, pH 7.5, and eluted with 0.12 M NaCl. Fab fragments were prepared by reduction and alkylation of F(ab')2 fragments using 0.1 M cysteine base in 0.1 M TrisHCI, pH 7.5, and 0.1 M iodoacetamide in 0.1 M Tris-HCI, pH 7.5, then fractionated by gel filtration on Sephadex G-25 superfine beads (Pharmacia, Piscataway, NJ) as described (22). The Ig concentration was measured by the method of Lowry and colleagues (23). The Ftab'), and Fab proteolytic fragments were examined for contamination by undigested anti-RMA by SDS-PAGE and silver staining. This technique detects 1 to 5 ng/ml of mAb (24) but failed to show the presence of conformationally intact anti-RMA in the proteolytic fragment preparations. Anti-RMA, F(ab')2' and Fab were diluted to 6.25 X 10-9 M in complete medium prior to their use with AM. Thymidine Incorporation and Cell Proliferation Assays AM (1 x lOS/well) in RPMI-1640 with 5 % FCS were plated in 96-well, flat-bottom microtiter culture dishes (Falcon), allowed to adhere for 1 h at 37° C, and wells were washed 3 times with warm PBS to remove nonadherent cells. Adherent AM were treated with dilutions of anti-RMA (0.01 ng/ml to 10 #Lg/ml), OX41 supernatant (""25 #Lg/ml; a kind gift of Dr. A. F. Williams, Oxford, UK) (25), phorbol myristate acetate (PMA; Sigma Chemical Co., St. Louis, MO; used at 10-7 to 10- 10 M), murine interferon-gamma (IFN--y; Genentech, S. San Francisco, CA; 0.1 to 10 U/ml) , lipopolysaccharide (LPS; Sigma; 0.1 to 10 #Lg/ml), or Fab and F(ab')2 fragments of anti-RMA (6.25 x 10-9 M). In some experiments, antiRMA (0.1 to 10 #Lg/ml) was added to PMA (10-10 M) or IFN-,.. (1 U/ml). To assess (3H]thymidine incorporation, AM were cultured in complete medium for 48 h, 1 #LCi of (3H]thymidine (NEN Research Products, Boston, MA; sp act, 84 Ci/mmol) was added per well, and the culture was incubated for 20 h. A total of 50 #LI of 1 M NaOH was added to each well for 10 min to solubilize the cells. The cells were harvested on a semi-automated cell harvester (Skatron, Sterling, VA), and the filters dried overnight at 37° C and counted in a {j-spectrometer (Packard, Sterling, VA). To assess parallel changes in total AM number, 0.5 X 106 AM were plated into 24-well culture dishes for 24 to 96 h. AM were rendered nonadherent by treatment with lidocaine (2 mg/ml) for 15 min, and counted in a hemocytometer. PH]thymidine Autoradiography AM (1 x 106/well) were plated in single-chamber glass slides (Lab-tek; Miles Laboratories, Napersville, IL), and nonadherent cells were removed by washing after a l-h incubation at 37° C.I Anti-RMA, anti-W3/25 (a murine anti-rat CD4 mAb) (26), or medium alone were added, and the cells were cultured for 24 h. (3H]thymidine (5 #LCi/well; NEN

Research Products) was added for 6 h, and the slides were washed 3 times in PBS, air-dried, and fixed in acetone for 5 min at room temperature. Slides were dipped in NTB-2 photographic emulsion (Eastman Kodak Co., Rochester, NY) and exposed for 2 wk at 4°C. Emulsion-coated slides were developed, counterstained with hematoxylin and eosin, and mounted with Permount (Fischer Scientific, Fairlawn, NJ). Assays for Multinucleated Cell Formation Glass coverslips (13 mm; Bellco Biotechnology, Vineland, NJ) were prepared by immersion in absolute ethanol for 60 min, washed 10 times in distilled water, air-dried, and sterilized by autoclaving. AM (5 x lOS/well) were plated onto the coverslips in 24-well culture dishes (Falcon) and allowed to adhere for 2 h at 37° C. Nonadherent cells were removed, and medium containing anti-RMA (0.0001 to 10 #Lg/ml), PMA (10-7 M), IFN-,.. (10 U/ml), LPS (0.01 to 10 #Lg/ml), or Fab or F(ab')2 fragments of anti-RMA (6.25 X 10- 9 M) added. After 5 d of culture at 37° C in 5 % CO 2, AM were washed 3 times in warm PBS and fixed for 10 min in 1% paraformaldehyde/PBS. The coverslips were removed, stained with 1% toluidine blue, and mounted on glass slides with Permount (Fischer Scientific). A minimum of 200 cells were randomly counted by light microscopy using a 40x objective and a lOx calibrated ocular. The percentage of multinucleated cells was calculated as the (number of AM containing > 1 nucleus/total number of AM counted) X 100. Electron Microscopy AM (1 X lOS) were plated in wells of an 8-chamber glass slide (Lab-tek; Miles Laboratories), incubated for 72 h with anti-RMA (5 #Lg/ml), OX41 (""25 #Lg/ml), or medium controls. Cells were fixed for 45 min with 2 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, washed with 0.15 M sodium cacodylate buffer, pH 7.4, and postfixed for 45 min in 1. 3 % Os04/s-collidine buffer. After washing with 0.15 M sodium cacodylate, AM were stained en bloc with 3% uranyl acetate in 75% ethanol, and dehydrated with graded ethanol. The culture chambers were filled with Epon and polymerized at 60° C overnight. Thin sections were stained with lead citrate and examined in a Philips 301 electron microscope (Philips Corp., Einthoven, The Netherlands). Statistical Methods Data were analyzed using an unpaired t test or a one-way analysis of variance with the Fischer PLSD test or Scheffe's F test for multiple comparisons, when appropriate, with a level of significance of P < 0.05. Data are expressed as the mean ± SEM.

Results AM Treated with Anti-RMA Display Increased MOC Formation Anti-RMA (IgG1K) induced homotypic cluster formation by AM isolated by saline lavage. By contrast, AM cultured with medium alone or with OX41 (IgG2a), a mAb that binds to > 90% of rat AM, did not form clusters (Figure 1). The number of AM involved in clustering reached a maximum at 24 h. The F(ab')2fragments of anti-RMA induced cluster

Figure 1. Clustering of AM induced by anti-RMA. AM (1 x lOS) were plated onto 96-well microtiter culture plates and incubated for 24 h in the presence of OX41 (25 jlg/ml) (upper panel) or anti-RMA (1 jlg/ml) (lower panel). Virtually all AM treated with anti-RMA are involved in cluster formation. (Magnification: x120.)

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formation by AM; Fab fragments of anti-RMA and the mAb W3/25 (IgG l ) (not shown) did not. A subset of AM stimulated with anti-RMA formed MGC (Figure 2). Saturating dosages (~ I ttg/ml) of anti-RMA (assessed by cytofluorimetry) produced MGC in 8.5 ± 0.5% of adherent AM at 48 h, and in 15.2 ± 0.6% of AM at 120 h (Figure 3). Only 2.4 ± 0.5 % of adherent AM cultured in medium and 3.6 ± 1.0% of adherent AM cultured with OX41 formed MGC at 120 h. PMA (10-1 M) produced MGC in 13.8 ± 1.8% of adherent AM. Combined stimulation with anti-RMA (I ttg/ml) and PMA (10-1 M) yielded MGC in

21.5 ± 6.5% of AM. The percentage ofMGC formed in the presence of anti-RMA varied with the concentration of the antibody (0 to 10 ttg/ml) (Figure 4). Morphology of MGC The MCG formed by anti-RMA shared morphologic features with giant cells in immune granulomas (27). MGC produced by anti-RMA contained as many as 12 nuclei/cell (Figure 5); most of these nuclei showed prominent nucleoli and sparse peripheral chromatin. Large numbers of irregularly shaped mitochondria and profiles of rough endoplasmic

Figure 2. MGC formation by AM incubated with anti-RMA . AM (5 X 1O~) were plated onto glass coverslips and incubated with medium alone (upper panel) or anti-RMA (1 j.tg/rnl) (lower panel) for 5 d, and stained. Note the presence of MGC formed by AM treated with antiRMA (arrows). Many of these cells developed a "foamy" appearance, demonstrated by electron microscopy to be due to dilated endoplasmic reticulum containing electron-dense material possibly reflecting increased protein synthesis following activation with anti-RMA. (Toluidine blue stain ; magnification: x313.)

Lazarus, Yamin, McCarthy et al.: Anti-RMA: Murine mAb that Activates Rat Macrophages

107

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(13). Both clustering (31) and polykaryon formation by monocytes (17) have been induced by IFN-"(, possibly due to the ability of this lymphokine to promote the activities of LFA-1 by these cells. Although a variety of antibodies can induce cluster formation by monocytes (32) , the murine anti-rat AM antibodies OX41 and W3/25 did not induce cluster formation in these studies. Clustering may potentially be achieved by antibody-mediated bridging of surface antigens to Fe receptors on adjacent macrophages (32). However, the observation that the F(ab)2fragment of anti-RMA also had the capacity to induce cluster formation suggests that binding of RMA antigens on adjacent AM by the antigen-combining domains ofthe antibody may promote cluster formation. Alternatively, cross-linking of adjacent RMA antigens on the surface of the same cell may induce secondary expression of surface adhesion molecules, such as LFA-I,or alter the activity of these molecules (33), leading to increased adhesion and cluster formation . Unfortunately, as specific mAbs against LFA-1 molecules in the rat were not available for these studies, we were unable to investigate the latter possibility directly. The finding that anti-RMA and its Ftab'), proteolytic fragment both induced MOC formation, confirms that the functional effects of anti-RMA do not result from nonspecific binding to the Fe receptors of AM .

Mariano and Spector (20) have reported an increase in DNA synthesis by macrophages participating in MOC formation. AM incubated with anti-RMA for 72 h displayed a marked increase in pH]thymidine incorporation. The heterogeneous radiolabeling of nuclei within MOC, and the failure to demonstrate a significant increase in the number of AM following stimulation with anti-RMA, suggests that DNA synthesis may be accompanied by fusion of AM and MOC formation . Although AM have generally been reported to display a limited capacity to synthesize DNA (34-39) , the degree of pH]thymidine incorporation induced by anti-RMA is consistent with other studies demonstrating that AM can proliferate in situ (40, 41). Several of the functional activities produced by anti-RMA were compared to those produced by PMA. PMA, a phorbol ester, activates protein kinase C directly, leading to the expression of competence genes (42-48) and DNA synthesis (43,49,50). Like anti-RMA, PMA induced both DNA synthesis and MOC formation by AM . Anti-RMA increa sed the percentage of MOC produced by PMA but also partially inhibited the level of (3H]thyrnidine incorporation produced by PMA. The cause of these differences in the effects of antiRMA on the activities induced by PMA is uncertain, but the results suggest that they may take place at different subcellular levels, i.e., nuclear versus surface membrane.

Lazarus, Yamin, McCarthy et al.: Anti-RMA: Murine mAb that Activates Rat Macrophages

In summary, anti-RMA promotes clustering, DNA synthesis, and MGC formation by AM. The possibility that antiRMA mimics a naturally occurring ligand that can bind to the RMA antigen and activate AM in vivo is being investigated. Acknowledgments: We gratefully acknowledge the technical assistance of Caryl Dailey in the development and screening of the hybridomas and the performance of some of these experiments. This study was supported by Grant HL36781 from the National Institutes of Health. DL was supported by Training Grant T32CA09216 from the National Institutes of Health.

References 1. Adams, D.O., and T. A. Hamilton. 1984. The cell biology of macrophage activation. Annu. Rev. Immuno!. 2:283. 2. Fels, A. 0., and Z. A. Cohn. 1986. The alveolar macrophage. J. Appl. Physiol. 60:353. 3. Allen, P. M., and E. R. Unanue. 1984. Differential requirements for antigen processing by macrophages for lysozyme-specific T cell hybridomas. J. Immunol. 132: 1077. 4. Brain, J. 1985. Macrophages in the respiratory tract. In The Handbook of Physiology. Section 3. The Respiratory System. Vol. 1. Circulation and Nonrespiratory Functions. A. P. Fishman, editor. American Physiological Society, Bethesda, MD. 447-471. 5. Papadimitriou, J., M. Sforsina, and L. Papaelias. 1973. Kinetics of multinucleated giant cell formation and their modification by various agents in foreign body reactions. Am. J. Pathol. 73:349. 6. Sutton, J., and L. Weiss. 1966. Transformation of monocytes in tissue culture into macrophages, epithelial cells and multinucleated giant cells. An electron microscope study. J. Cell BioI. 28:303. 7. Black, M., and W. Epstein. 1974. Formation of multinucleated giant cells in organized epithelioid cell granulomas. Am. J. Pathol. 74:263. 8. Galindo, B. 1972. Antigen-mediated fusion of specifically sensitized rabbit alveolar macrophages. Infect. Immun. 5:583. 9. Postlethwaite, A., B. Jackson, E. Beachey, and A. Kang. 1982. Formation of multinucleated giant cells from human monocyte precursors. J. Exp. Med. 155:168. 10. Schlesinger, L., R. Musson, and R. Johnston. 1984. Functional and biochemical studies of multinucleated giant cells derived from cultured human monocytes. J. Exp. Med. 159: 1289. 11. Chambers, T. J. 1978. Multinucleate giant cells. J. Patho!' 126:125. 12. Kawanami, 0., F. Basset, R. Barrios, J. G. Lacronique, V. J. Ferrans, and R. G. Crystal. 1983. Hypersensitivity pneumonitis in man. Am. J. Pathol. 110:275. 13. Sone, S., C. Bucana, L. C. Hoyer, and I. J. Fidler. 1981. Kinetics and ultrastructural studies of the induction of rat alveolar macrophage fusion by mediators released from mitogen-stimulated lymphocytes. Am. J. Pathol. 103:234. 14. Boros, D. L. 1981. The role of lymphokines in granulomatous inflammations. In Lymphokines. E. Pick, editor. Academic Press, New York. 3:257. 15. Galindo, B., J. Lazdins, and R. Castillo. 1974. Fusion of normal rabbit alveolar macrophages induced by supernatant fluids from BCG-sensitized lymph node cells after elicitation by antigen. Infect. Immun. 9:212. 16. Screiber, R. D., J. L. Pace, S. W. Russell, A. Altman, and D. H. Katz. 1983. Macrophage activating factor produced by aT-cell hybridoma: physiochemical and biosynthetic resemblance to ')'-interferon. J. Immunol. 131:826. 17. Weinberg, 1. B., M. M. Hobbs, and M. A. Misukonis. 1985. Phenotypic characterization of gamma interferon-induced human monocyte polykaryons. Blood 66:1241. 18. McInnes A., and D. M. Rennick. 1988. Interleukin 4 induces cultured monocytes/macrophages to form multinucleated cells. J. Exp. Med. 167:598. 19. Abe, E., Y Ishimi, H. Tanaka et al. 1987. The relationship between fusion and proliferation in mouse alveolar macrophages. Endocrinology 121:271. 20. Mariano, M., and W. G. Spector. 1974. The formation and properties of macrophage polykaryons (inflammatory giant cells). J. Pathol. 113:1. 21. Yamin, Y, D. Lazarus, E. E. Schneeberger, K. McCarthy, W. Xia, and R. Kradin. 1989. Anti-RMA: a murine monoclonal antibody that activates rat macrophages. I. Distribution and characterization of the RMA antigen. Am. J. Respir. Cell. Mol. BioI. 2:207. 22. Parham, P. 1986. Preparation and purification of active fragments from mouse monoclonal antibodies. In Handbook of Experimental Immunology. Vol. 1. Immunochemistry. Chapter 14. D. M. Weir, L. A. Herzenberg, C. Blackwell, and L. A. Herzenberg, editors. Blackwell Scientific Publications, London. 23. Lowry, D.'H., N. 1. Rosenborough, A. L. Farr, and R. 1. Randall. 1951. Protein measurement with folin phenol reagent. J. Biol. Chern. 193:265. 24. Oakley, B. R., D. R. Kirsch, and N. R. Morris. 1980. A simplified ultrasen-

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sitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105:361. 25. Robinson, A. P., T. M. White, and 0. W. Mason. 1986. Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies, MRC OX41 and MRC OX 42, the latter recognizing complement receptor type 3. Immunology 57:239. 26. Williams, A. F., G. Galfre, and C. Millstein. 1977. Analysis of cell surfaces by xenographic myeloma-hybrid antibodies. Differentiation of rat lymphocytes. Cell 12:633. 27. Williams, G. T., and W. J. Williams. 1983. Granulomatous inflammation-a review. J. Clin. Pathol. 36:723. 28. Robinson, S. H., G. Brecher, S. I. Lourie, and J. E. Haley. 1965. Leukocyte labeling in rats during and after continuous infusion oftritated thymidine: implications for lymphocyte longevity and DNA reutilisation. Blood 26:281. 29. Inaba, K., and R. M. Steinman. 1986. Accessory cell-T lymphocyte interactions. Antigen-dependent and -independent clustering. J. Exp. Med. 163:247. 30. Kradin, R. L., K. M. McCarthy, C. I. Dailey, A. Burdeshaw, J. T. Kurnick, and E. E. Schneeberger. 1987. The poor accessory cell function of macrophages in the rat may reflect their inability to form clusters with T cells. Clin. Immunol. Immunopathol. 44:22. 31. Mentzer, S. J., D. V. Faller, and S. J. Burakoff. 1986. Interferon-y induction of LFA-l mediated homotypic adhesion of human monocytes. J. Immunol. 137:108. 32. Strussman, G., T. A. Springer, and 0. 0. Adams. 1985. Studies on antigens associated with the activation of murine mononuclear phagocytes: kinetics of the requirements for induction oflymphocyte function-associated (LFA1) antigen in vitro. J. Immunol. 135:147. 33. Wawryk, S. 0.,1. R. Novotny, I. P. Wicks et al. 1989. The role of the LFAl/lCAM interaction in human leukocyte homing and adhesion. Immunol. Rev. 108:135. 34. Blusse van oud Ablas, A., and R. van Furth. 1979. Origin, kinetics and characteristics of pulmonary macrophages in the normal steady state. J. Exp. Med. 149:1504. 35. Blusse van oud Ablas, A., B. van der Linder-Schrerier, and R. van Furth. 1983. Origin and kinetics of pulmonary macrophages during an inflammatory reaction induced by intra-alveolar administration of aerosolized, heatkilled BCG. Am. Rev. Respir. Dis. 128:276. 36. Adamson, I., and P. Bowden. 1980. Role of monocytes and interstitial cells in the generation of alveolar macrophages: kinetic studies after carbon loading. Lab. Invest. 42:518. 37. Bitterman, P., L. Saltzman, S. Adelberg, V. Ferrans, and R. Crystal. 1984. Alveolar macrophage replication: one mechanism for the expansion of the mononuclear phagocyte population in the chronically inflamed lung. J. cu« Invest. 74:460. 38. Metcalf, D. 1982. Regulation of macrophage production. Adv. Exp. BioI. Med. 155:33. 39. Golde, D., T. Finley, and M. Cline. 1974. The pulmonary macrophage in acute leukemia. N. Engl. 1. Med. 290:875. 40. Thrling, 1. D., H.-S. Lin, and S. Hsu. 1987. Self-renewal of pulmonary alveolar macrophages: evidence from radiation chimera studies. J. Leukocyte Biol. 42:443. 41. Shellito, 1., C. Esparza, and C. Armstrong. 1987. Maintenance of normal rat alveolar macrophage cell population. The roles of monocyte influx and alveolar macrophage proliferation in situ. Am. Rev. Respir: Dis. 135:78. 42. Geppert, T. D., and P. E. Lipsky. 1987. Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3. J. Immunol. 138:1660. 43. Abdel-Latif, A. A. 1986. Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol. Rev. 38:227. 44. Fujita, I., K. Irita, K. Takeshige, and S. Minakami. 1984. Diacylglycerol l-oleoyl-z-acetyl-glycerol stimulates superoxide generation from human neutrophils. Biochem. Biophys. Res. Commun. 120:312. 45. Mastro, A. M. 1982. Phorbol esters: tumor promotion, cell regulation and the immune system, In Lymphokines. Vol. 6. S. B. Mizel, editor. Academic Press, New York. 263-313. 46. Cochrane, B. H. 1985. The molecular action of platelet-derived growth factor. Adv. Cancer Res. 45:183. 47. Introna, M., T. A. Hamilton, R. E. Kaufman, D. O. Adams, and R. C. Bast. 1986. Treatment of murine peritoneal macrophages with bacteriallipopolysaccharide alters expresion of c-fos and c-myc oncogenes. J. Immunol. 137:2711. 48. Coughlin, S. R., W. M. F. Lee, P. W. Williams, G. M. Giels, and L. T. Williams. 1985. c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF. Cell 43:243. 49. Dicker, P., and E. Rozengurt. 1980. Phorbol esters and vasopressin stimulate DNA synthesis by a common pathway. Nature 287:607. 50. Rozengurt, E., A. Rodriquez-Pena, M. Coombs, and 1. Sinnett-Smith. 1984. Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells: role of Ca 2 + -sensitive phospholipid-dependent protein kinase. Proc. Natl. Acad. Sci. USA 81:5748.