(tumor immunology/neutrophil/13762NF rat mammary adenocarcinoma). DANNY R. WELCH*t*, DANIEL J. SCHISSEL*, RICHARD P. HOWREY*, AND PAUL A.
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 5859-5863, August 1989
Cell Biology
Tumor-elicited polymorphonuclear cells, in contrast to "normal" circulating polymorphonuclear cells, stimulate invasive and metastatic potentials of rat mammary adenocarcinoma cells (tumor immunology/neutrophil/13762NF rat mammary adenocarcinoma)
DANNY R. WELCH*t*, DANIEL J. SCHISSEL*, RICHARD P. HOWREY*, AND PAUL A. AEEDt *Division of Chemotherapy, Glaxo Research Laboratories, Research Triangle Park, NC 27709; and tDepartment of Cancer and Infectious Diseases Research, The Upjohn Company, Kalamazoo, MI 49001
Communicated by Pedro Cuatrecasas, May 12, 1989 (received for review February 6, 1989)
ABSTRACT Circulating polymorphonuclear cell (PMN) levels rise in proportion to the metastatic potential of the tumor in 13762NF mammary adenocarcinoma tumor-bearing rats. These tumor-elicited PMNs (tcPMNs) secrete high levels of the basement-membrane-degrading enzymes, type IV collagenase and heparanase, suggesting that metastatic tumor cells stimulate neutrophilia so that the tcPMNs might assist tumor cell extravasation during metastasis. To test this hypothesis, purified proteose peptone-elicited PMNs from peritoneal exudate, circulating normal PMNs, and tcPMNs were evaluated for their effects on in vitro invasive and in vivo metastatic potentials of syngeneic 13762NF mammary adenocarcinoma tumor cells. tcPMNs caused a dose-dependent increase in invasion through a reconstituted basement membrane barrier in an in vitro invasion assay. At PMN:tumor cell ratios of 30:1, invasion potential significantly (P < 0.05) rose to 26-fold, 40-fold, and 37-fold for poorly metastatic MTLn2 cells, higly metastatic MTLn3 cells, and moderately metastatic MTF7 cells, respectively. In contrast, purified proteose peptone-elicited PMNs and circulating normal PMNs did not sgificantly alter invasive potential. Intravenous coij'ections of purified proteose peptone-elicited PMNs did not change the number of experimental lung metastases, but tcPMNs at ratios to 50:1 significantly raised the mean number of metasass 23-fold for MTLn2, 3- to 4-fold for MTLn3, and 1.6- to 1.8-fold for MTF7. These results demonstrate that tcPMNs contribute to the metastatic propensity of mammary adeocarcinoma clones by increasing efficiency of invasion through basement membrane.
hypothesized that immune cells could subsidize deficient properties of weakly metastatic populations within a heterogeneous tumor, thus giving these tumor cells the ability to metastasize. Also, immune cells could increase metastatic efficiency of tumor cells. Others have previously shown that lymphocytes (12, 13), mast cells (14, 15), and macrophages (16) can enhance metastatic potential in various animal tumors. Although polymorphonuclear cells (PMNs) are not the predominant circulating leukocyte population in normal rats, PMNs are the predominant population in humans. Neutrophils have been seen in close association with metastatic human and animal tumor cells in vivo at the primary tumor (10) and within the vasculature (17). The observation that the level of circulating PMNs increases to 50-fold as the primary tumor proliferates and the observation that tumor-elicited PMNs (tcPMNs) secrete high levels of type IV collagenase and heparanase and are noncytotoxic and noncytostatic (9), combine to suggest that tcPMNs may enhance the ability of tumor cells to extravasate, hence to metastasize. The results presented here demonstrate that tcPMNs, but not normal circulating PMNs (cPMNs), augment the ability of a tumor cell to penetrate a basement membrane-like matrix in vitro and to form lung colonies in vivo.
MATERIALS AND METHODS Animals. Pathogen- and virus-free, 6- to 7-week old female Fischer 344/NHSd (F344) rats were obtained from Harlan Sprague Dawley. Animals were maintained under the guidelines of Glaxo, Upjohn, and the National Institutes of Health. Rats were fed Purina rodent chow and tap water (chlorine content was 0.05) by one-way analysis of variance. MTF7 metastatic potential was also unchanged when pp-
Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Effect of ppPMNs on metastatic potential of 13762NF rat mammary adenocarcinoma cells Relative metastatic Lung metastases, no. potential Treatment Cell line 1.00 31 ± 6 Tumor cells MTF7 (T19) 1.38 43 ± 7 + ppPMN (10") + ppPMN (105) 0.91 28 ± 4 + ppPMN (106) 0.79 24 ± 5 1.00 39 ± 2 MTLn3 (T48) Tumor cells + ppPMN (10") 1.06 41 ± 3 + ppPMN (105) 1.10 43 ± 2 + ppPMN (106) 0.82 32 ± 7 Viable tumor cells (105 cells per 0.2 ml) were injected into the lateral tail veins of F344/NHSd female rats. Animals were sacrificed 14 days postinjection, lungs were removed, and the number of surface lung metastases was counted with the aid of a dissecting microscope. Data are presented as mean ± SEM. One-way analysis of variance was done using the Abstat statistical package to compare groups. Relative metastatic potential was calculated as the ratio of ppPMN-treated vs. untreated tumor cell metastic potential within the same experiment (n 2 10); ppPMNs were isolated from peritoneal exudate of rats injected 4-6 hr previously with a 10%6 sterile proteose peptone solution.
PMNs were coinjected (range 24-43). Similarly, 13762NF mammary adenocarcinoma cells injected after growth for 24-72 hr in ppPMN-conditioned medium did not exhibit altered metastatic potentials (data not shown). ppPMNs also did not significantly (P > 0.05) alter relative invasive potentials of MTLn2, MTF7, or MTLn3 cells in MICS (Table 2). cPMNs produced only nominal effects on relative invasive potentials of MTLn3 cells (Table 2). cPMNs Table 2. Effect of "normal" PMNs on invasion of 13762NF rat mammary adenocarcinoma cells in the MICS Relative invasive potential Treatment Invasion, % Cell line 1.00 0.54 ± 0.004 MTLn2 (T41) Tumor cells + ppPMN (105) 1.20 0.65 ± 0.008 1.52 + ppPMN (106) 0.82 ± 0.007 1.00 0.04 ± 0.001 MTLn2 (T43) Tumor cells 1.25 + ppPMN (105) 0.05 ± 0.001 1.50 + ppPMN (106) 0.06 ± 0.001 1.00 0.45 ± 0.005 MTLn3 (T53) Tumor cells ND + ppPMN (105) ND + ppPMN (106) 1.10 0.49 ± 0.009 1.00 1.35 ± 0.059 MTLn3 (T56) Tumor cells + ppPMN (105) 0.90 1.22 ± 0.038 + ppPMN (106) 0.91 1.23 ± 0.047 1.00 0.55 ± 0.013 Tumor cells MTLn3 (T54) + cPMN (105) 0.91 0.51 ± 0.004 1.17 + cPMN (106) 0.65 ± 0.008 1.00 MTF7 (T17) Tumor cells 1.07 ± 0.010 + ppPMN (106) 0.96 1.01 ± 0.010 + ppPMN (106) 1.09 1.17 ± 0.020 1.00 0.67 ± 0.041 MTF7 (T19) Tumor cells 0.94 + ppPMN (105) 0.63 ± 0.028 1.04 + ppPMN (106) 0.70 ± 0.035 Tumor cells were seeded at 105 (in 1.5 ml) per well in the MICS chamber. Chambers were incubated for 72 hr, medium containing invading cells was removed through side-sampling ports, and cells were isolated by filtering them onto a 0.45-,um filter with a Minifold filtration apparatus. Retained cells were fixed, stained, and counted as described (19). Percent invasion is expressed as mean ± SEM. Relative invasive potential was calculated as the ratio of ppPMNtreated vs. untreated tumor cells within the same experiment (n = 4). No significant differences were found by analysis of variance. ND, not determined.
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were included in these studies to assure that the source of normal PMNs was not a major variable in these studies. Wells receiving cPMNs or ppPMNs with tumor cells generated a relative invasive potential of 0.90-1.17, except for MTLn2 cells. MTLn2 invasive potential exhibited a dose-dependent increase to 1.50, but these differences were not statistically significant. Addition of phorbol 12-myristate-13-acetate (10 ng/ml), a potent activator of neutrophils, did not alter invasion through reconstituted basement membrane in MICS (data not shown). For all experiments, percent invasion was normalized to cell invasion of the untreated control set at 100%; this was done because actual percent invasion varied between experiments, mostly as a result of the Matrigel thickness (Table 2). Effect of tcPMNs on Invasion and Experimental Metastasis. The effect of tcPMNs on the ability of MTLn2, MTLn3, and MTF7 clones to penetrate reconstituted basement membrane was measured in the MICS assay at ratios of 3:1 and 30:1. A dose-dependent rise in relative invasive potentials was seen for all cell lines (Table 3). MTLn3 tumor-cell relative invasive potential increased 12- to 40-fold at tcPMN:tumor cell ratios of 30:1. Wells seeded with MTLn2 cells gave invasive potentials up to 7-fold and 25.5-fold higher for PMN:tumor cell ratios of 3:1 and 30:1, respectively. MTF7 relative invasive potentials rose to 14-fold and 36-fold with PMN:tumor cell ratios of 3:1 and 30:1, respectively. These increases were highly statistically significant (P < 0.0001) despite variability in absolute percent invasion in individual experiments. The magnitude of tcPMN enhancement of invasion potential was less when tumor cells themselves penetrated more easily (due to a thinner Matrigel barrier) in control wells. There was always an enhancement of invasive and metastatic potential for all experiments. Coinjection of tumor cells of varying metastatic potential with neutrophils isolated from MTLn3 tumor-bearing rats Table 3. Effect of tcPMNs on invasive potential of 13762NF mammary adenocarcinoma cells in the MICS Relative invasive Cell line Treatment Invasion, % potential MTLn2 (T38) Tumor cells 0.04 ± 0.001 1.00 + tcPMN (3 x 105) 0.27 ± 0.002 6.75* + tcPMN (3 x 106) 0.98 ± 0.008 25.50* MTLn2 (T40) Tumor cells 0.03 ± 0.005 1.00 + tcPMN (3 x 105) 0.08 ± 0.012 2.67* + tcPMN (3 x 106) 0.46 ± 0.054 15.33* MTLn3 (T49) Tumor cells 0.04 ± 0.050 1.00 + tcPMN (3 x 105) 0.38 ± 0.014 9.50* + tcPMN (3 x 106) 1.61 ± 0.058 40.25* MTLn3 (T51) Tumor cells 0.14 ± 0.014 1.00 + tcPMN (3 x 105) 0.39 ± 0.024 2.79* + tcPMN (3 x 106) 1.71 ± 0.068 12.21* MTF7 (T14) Tumor cells 2.81 ± 0.180 1.00 + tcPMN (3 x 105) 4.49 ± 0.130 1.60* + tcPMN (3 x 106) 11.47 ± 0.210 4.08* MTF7 (T16) Tumor cells 0.03 ± 0.005 1.00 + tcPMN (3 x 105) 0.42 ± 0.021 14.00* + tcPMN (3 x 106) 1.10 ± 0.050 36.67* Tumor cells were seeded at 105 (in 1.5 ml) per well in the MICS chamber. Chambers were incubated for 72 hr, medium containing invading cells was removed by means of side-sampling ports, and cells were isolated by filtering onto a 0.45-gm filter with a Minifold filtration apparatus. Retained cells were fixed, stained, and counted as described (19). Percent invasion is expressed as mean ± SEM. Relative invasive potential was calculated as the ratio of tcPMNtreated vs. untreated tumor cells within the same experiment (n = 4). tcPMNs were isolated from peripheral blood of 20- to 24-day-old MTLn3 tumor-bearing rats. *Significantly different from control (P < 0.0001).
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caused a dose-dependent increase in the number of experimental lung metastases for each tumor cell line (Table 4). Rats injected with MTLn3 cells and tcPMNs produced -2fold more lung metastases with tcPMN:tumor cell ratios of 5:1, and 3- to 4-fold more metastases with ratios of 50:1 (P < 0.005). The coinjection of tcPMNs with MTLn2 or MTF7 cells also increased the number of experimental lung metastases formed compared to an injection of MTLn2 or MTF7 cells alone. Coinjection with tcPMNs at ratios of 5:1 and 50:1 with poorly metastatic MTLn2 cells generated 5.36-fold and 23-fold more lung metastases, respectively. When MTF7 cells were coinjected with 5:1 tcPMN the number of metastases was not significantly different from control tumor cells, but when coinjected with 50:1 tcPMN, they formed 1.64- to 1.76-fold more lung colonies (P < 0.005). To rule out the possibility that the observed changes with tcPMN were due to contaminating host immune cells, smears of each preparation were examined microscopically. The purity of each preparation was always >99%.
DISCUSSION Only specialized subpopulations of tumor cells can perform all of the necessary steps in the complex metastatic cascade. Nonetheless, it is becoming apparent that tumor cells receive signals from their environment that can modulate metastatic potential (24-26). These signals come from either other tumor cells or the host. For example, tumor cells can collaborate to increase the efficiency of arrest in the microvasculature by forming homotypic emboli (27, 28) or heterotypic emboli with platelets and/or lymphocytes (1, 3, 29). Others (30, 31) have shown that the presence of neutrophils can enhance tumor cell adhesion to intimal components. The mechanisms invoived are largely unknown except that neutrophil-mediated endothelial damage by means of free radical production is thought to play a major role (31). We previously found that neutrophilia in 13762NF mammary adenocarcinoma tumor-bearing rats was proportional Table 4. Effect of tcPMNs on the metastatic potential of 13762NF mammary adenocarcinoma cell clones Relative metastatic Lung Cell line Treatment metastases, no. potential MTLn2 (T40) Tumor cells 1.00 0.56 ± 0.11 + tcPMN (5 x 105) 3 ± 1 5.36 + tcPMN (5 x 106) 11 ± 2 23.21* 1.00 MTLn3 (T48) Tumor cells 26 ± 1 + tcPMN (5 x 105) 54 ± 4 2.08* + tcPMN (5 x 106) 76 ± 5 3.04* 1.00 MTLn3 (T51) Tumor cells 39 ± 4 + tcPMN (5 x 105) 2.23* 87 ± 7 + tcPMN (5 x 106) 4.00* 156 ± 12 MTF7 (T19) Tumor cells 55 ± 2 1.00 + tcPMN (5 x 105) 1.27 70 ± 3 + tcPMN (5 x 106) 1.64* 90 ± 4 MTF7 (T19) Tumor cells 1.00 46 ± 5 1.15 + tcPMN (5 x 105) 53 ± 4 + tcPMN (5 x 106) 81 ± 6 1.76* Viable tumor cells (105 cells per 0.2 ml) were injected into the lateral tail veins of F344/NHSd female rats. Animals were sacrificed 14-18 days postinoculation, lungs were removed, and the number of surface lung metastases were counted with the aid of a dissecting microscope. Lung metastases are reported as mean ± SEM. Analysis of variance was done using the Abstat statistical package. Relative metastasic potential was calculated as the ratio of tcPMNtreated vs. untreated tumor cells within the same experiment (n 2 10). tcPMNs were isolated from peripheral blood of 20- to 24-day-old MTLn3 tumor-bearing rats. *Significantly different from control (P < 0.005).
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to the metastatic potential of the tumor population (9). That tcPMN, cPMN, and ppPMN were neither cytotoxic nor cytostatic and that tcPMN secreted -50% more type IV collagenase and heparanase than cPMN suggested that neutrophils from tumor-bearing rats were helping, rather than inhibiting, 13762NF mammary adenocarcinoma cell metastatic potential by assisting extravasation. How neutrophilia is induced by the malignant subpopulations of a heterogeneous tumor is still uncertain, although granulocyte-monocyte colony-stimulating factor production has been implicated in some tumor systems (32). Our results indicate that mere stimulation of granulocyte-monocyte differentiation is not all that is occurring. Not only are circulating PMN numbers rising, but each tcPMN produces higher-than-normal levels of proteinases. Further, activation of either ppPMN or cPMN with phorbol 12-myristate-13-acetate at 10 ng/ml did not increase invasion in MICS; neither was experimental metastatic potential altered (unpublished observations), indicating that activation of PMN was occurring at a higher level and that tumor cells were providing the activating signal. It remains to be determined whether tcPMNs are equally "activated" by less malignant tumors and whether tcPMNs elicited by different tumors exert the same effects on metastatic potential for all tumor subpopulations. tcPMNs significantly enhanced the ability of 13762NF mammary adenocarcinoma tumor cells to invade reconstituted basement membrane barriers by up to 40-fold at ratios of 30:1 and increased experimental metastatic potential from 2- to 23-fold at ratios of 50:1. The magnitude of enhancement is even more astonishing because the number of circulating tcPMNs to tumor cells in vivo could be 500:1 when 100,000 cells are in the vasculature (9). The reasons for a lower enhancement ratio of experimental metastatic potential compared with invasion potential are several fold. (i) In MICS the tumor cells are continuously exposed to the tcPMNs for 72 hr. Although the kinetics of invasion in vivo is similar, it is possible that tcPMNs are not in close association with tumor cells continuously for that period of time. On the other hand, Crissman et al. (17) did find close association for long periods (>48 hr) in vivo. (ii) More importantly, any in vitro assay elminates many of the other selective pressures (variables) imposed on tumor cells during the metastatic cascade. Though more efficient at the extravasation step, tumor cells may still be susceptible to killing at other points. (iii) The basement membrane forms a significantly more complex barrier than filters coated with reconstituted, tumor-derived basement membrane extract. Also, the composition of the basement membrane in vivo is different in that it contains different structural components as well as tissue growth factors (33). These growth factors could be either stimulatory or inhibitory (34, 35) and could be released upon basement membrane degradation (33). Though analogous, we acknowledge differences between the in vivo and the in vitro barriers that could account for the magnitude of the observations described. (iv) 13762NF mammary adenocarcinoma cells are particularly susceptible to motility factors (36) and the enhancement in vitro could be partially due to secretion of motility factors that would obviously increase invasion. Likewise, we cannot rule out that tcPMNs may contribute to malignancy by enhancing other steps in the metastatic process, such as attachment, aggregation, or growth potential. Although it has been speculated for many years that immune cells could assist tumor cell invasion, direct evidence is minimal. Others have shown that macrophages, lymphocytes, mast cells, and fibroblasts can be induced by tumor cells to secrete increased amounts of proteolytic enzymes that presumably assist invasion and metastasis (14-16, 26, 37). Although we do not have direct in vivo evidence for PMN involvement in 13762NF mammary adenocarcinoma extravasation, the data presented here do confirm increased
Proc. Natl. Acad. Sci. USA 86 (1989)
invasion, albeit in vitro, and metastasis caused by immune cell populations. Perhaps the most important finding is that poorly metastatic cells can be made much more malignant when coinjected with tcPMNs. This suggests that metastatic populations can elicit neutrophilia (which is specialized in that the tcPMNs are highly activated) and that nonmetastatic or poorly metastatic subpopulations within the same tumor can take advantage of the opportunistic environment provided by the tcPMN to increase their apparent metastatic potential. This finding, besides providing an explanation for finding nonmetastatic cell populations within individual tumor metastases, also indicates the potential for developing antimetastatic therapy to an accessory cell, in this case the tcPMN. We thank Drs. Thea Tlsty and Richard Seftor for their critical
reading of this manuscript and helpful suggestions. 1. Nicolson, G. L. (1988) Biochim. Biophys. Acta 948, 175-224. 2. Fidler, I. J. & Nicholson, G. L. (1987) Cancer Bull. 39, 126131. 3. Fidler, I. J. & Kripke, M. L. (1980) Cancer Immunol. Immunother. 7, 201-205. 4. Poste, G. & Nicolson, G. L. (1983) Curr. Prob. Cancer 7, 1-42. 5. Hoover, R. I., Folger, R., Hering, W. A., Ware, B. R. & Karnovsky, M. J. (1980) J. Cell Sci. 45, 73-86. 6. Terranova, V. P., DiFlorio, R., Hujanan, E. S., Lyall, R. H., Liotta, L. A., Thorgiersson, U. P., Siegal, S. P. & Schiffmann, E. (1986) J. Clin. Invest. 77, 1180-1186. 7. Zurier, R. B., Hoffstein, S. & Weissmann, G. (1973) J. Cell Biol. 58, 27-41. 8. Wright, D. G. & Gallin, J. I. (1979) J. Immunol. 123, 285-294. 9. Aeed, P. A., Nakajima, M. & Welch, D. R. (1988) Int. J. Cancer 42, 748-756. 10. Neri, A., Welch, D. R., Kawaguchi, T. & Nicolson, G. L. (1982) J. Natl. Cancer Inst. 68, 507-517. 11. Goldfarb, R. H. & Liotta, L. A. (1986) Semin. Thrombosis Hemostasis 12, 294-306. 12. Gorelik, E., Wiltrout, R. H., Copeland, D. & Herberman, R. B. (1985) Cancer Immunol. Immunother. 19, 35-42. 13. Fidler, I. J., Gersten, D. J. & Budmen, M. B. (1976) Cancer Res. 36, 3160-3165. 14. Dabbous, M. H., Walker, R., Haney, L., Carter, L. M., Nicolson, G. L. & Wooley, D. E. (1986) Br. J. Cancer 54, 459-465. 15. Dabbous, M. H., Wooley, D. E., Haney, L., Carter, L. M. & Nicolson, G. L. (1986) Clin. Exp. Metastasis 4, 141-152. 16. Dabbous, M. H., North, S. M., Haney, L. & Nicolson, G. L. (1988) Cancer Res. 48, 6832-6836. 17. Crissman, J. D., Hatfield, J., Schaldenbrand, M., Sloane, B. F. & Honn, K. V. (1985) Lab. Invest. 53, 470-478. 18. Welch, D. R., Neri, A. & Nicolson, G. L. (1983) Invasion Metastasis 3, 65-80. 19. Welch, D. R., Lobl, T. J., Seftor, E. A., Wack, P. J., Aeed, P. A., Yohem, K. H., Seftor, R. E. B. & Hendrix, M. J. C. (1989) Intl. J. Cancer 43, 449-457. 20. Hendrix, M. J. C., Seftor, E. A., Seftor, R. E. B. & Fidler, I. J. (1987) Cancer Lett. 38, 137-147. 21. Weissmann, G. (1978) Hosp. Pract. 13, 53-62. 22. Samuelsson, B. (1983) Science 220, 568-575. 23. Moser, R., Schleiffenbaum, B., Groscurth, P. & Fehr, J. (1989) J. Clin. Invest. 83, 444-455. 24. Miller, F. R. (1983) Invasion Metastasis 3, 234-242. 25. Welch, D. R., Aeed, P. A., Earhart, R. H., Schissel, D. J., Howrey, R. P. & Hendrix, M. J. C. (1989) Proc. Am. Assoc. Cancer Res. 30, 352. 26. Dabbous, M. H., Haney, L., Carter, L., Paul, A. K. & Reger, J. (1987) J. Cell. Biochem. 35, 333-344. 27. Liotta, L. A., Kleinerman, J. & Sardel, G. M. (1976) Cancer Res. 36, 889-894. 28. Updyke, T. V. & Nicolson, G. L. (1986) Clin. Exp. Metastasis 4, 273-284. 29. Mentor, D. G., Hatfield, J. S., Harkins, C., Sloane, B. F., Taylor, J. D., Crissman, J. D. & Honn, K. V. (1987) Clin. Exp. Metastasis 5, 65-78.
Cell Biology: Welch et al. 30. Starkey, J. R., Liggitt, H. D., Jones, W. & Hosick, H. L. (1984) Int. J. Cancer 34, 535-543. 31. Orr, F. W. & Warner, D. J. (1987) Invasion Metastasis 7, 183-196. 32. Lee, M. Y. & Baylink, D. J. (1983) Proc. Soc. Exp. Biol. 172, 424-429. 33. Vladovski, I., Folkman, J., Sullivan, R., Fridman, R., IshaiMichaeli, R., Sasse, J. & Klagsbrun, M. (1987) Proc. Nati. Acad. Sci. USA 84, 2292-2296. 34. Horak, E., Darling, D. L. & Tarin, D. (1986) J. Nati. Cancer Inst. 76, 913-922.
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35. Nicolson, G. L. & Dulski, K. M. (1986) Intl. J. Cancer 38, 289-294. 36. Atnip, K. D., Carter, L. M., Nicolson, G. L. & Dabbous, M. H. (1987) Biochem. Biophys. Res. Commun. 146, 996-1002. 37. Pauli, B. U. & Knudson, W. (1988) Hum. Pathol. 19, 628-639. 38. Hibbs, M. S., Hasty, K. A., Kang, A. H. & Mainardi, C. L. (1984) Coll. Rel. Res. 4, 467-477. 39. Schalwijk, J., van den Berg, W. B., van de Putte, L. B. & Joosten, L. A. (1986) Br. J. Exp. Pathol. 68, 81-88. 40. Weiss, S. J., Curnutte, J. T. & Regiani, S. (1986) J. Immunol. 136, 636-641.
