Antiinflammatory effects of neoplasms

50th FORUM

268

ed polymorphonuclear Int.

J. Cancer,

46,

IN IA4A4UNOI~OGY

leukocytes and macrophages. 533-538.

Mahoney, M.J. & Leighton, J. (1962), The inflammatory response to a foreign body within transplantable tumors. Cancer Res., 22, 334-338. Mantovani, A., Bottazzi, B., Colotta, F., Sozzani, S. 6r Ruco, L. (1992), The origin and function of tumorassociated macrophages. Itnntunol. Todo)), 13, 265-270. Mignatti, P. & Rifkin, D.B. (1993), Biology and biochemistry of proteinases in tumor invasion. Physiol. Rev., 73, 161-195. Milon, G. & Fauve, R.M. (1976), Reactions immunitaires de souris C57Bl/6 au tours du dCveloppement d’une greffe tumorale: le carcinome pulmonaire de Lewis. Ann. Immunol. (Inst. Pasteur), l27C, 615-685. Movat, H.Z. (1979), Kinins and the kinin system as inflammatory mediators, in “Chemical messengers of the inflammatory process” (J.C. Houck) (pp. 47-l 12). Elsevier/North-Holland Biomedical Press, Amsterdam. Pryor, W.A. (1976), in “Free radicals in biology” (p. 32), vol. I. Academic Press, New York, London.

Antiinflammatory

effects of neoplasms G.J.

Macronex,

Reich, R.E., Thompson, E.W., Iwamoto, Y., Martin, G.R., Deason, J.D., Fuller, G.C. & Miskin, R. (1988), Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase 1V on the invasion of basement membranes by metastatic cells. Cancer Res., 48, 3307-3312. Ryan, C. & Majno, G. (1977), Acute inflammation. A review. Amer. J. Path.. 86, 185-276. Samak, R.. Edelstein, R. &Israel, L. (1982), Immunosuppressive effect of acute-phase reactant proteins in vitro and its relevance to cancer. Cancer Immunol. I/nrnunoth., 13, 38-43. Srimal, S. & Nathan, C.F. (1990), Purification of macrophage deactivating factor. J. esp. Med., 171, 1347-1355. Strauch, L. (1972), The role of collagenases in tumour invasion, in “Tissue interactions in carcinogenesis” (D. Tarin) (pp. 399-433). Academic Press, London, New York. Whitworth, P., Pak, C., Esgro, J., Kleinerman, E. & Fidler, I. (1990), Macrophages and Cancer. Cancer Metastasis Rev., 8, 319-351.

Cianciolo

Research and Development, 120 Southcenter Morrisville,

NC 27560-9136

For more than three decades there has been considerable scientific interest in characterizing the nature of the defective antiinflammatory responses associated with neoplasms. As occurs in many different fields of scientific discovery and pursuit, the paths which various scientists have followed in their quests to characterize these defects have as often diverged as converged. And yet, although many unanswered questions remain, I feel that at least some of our initial questions are close to being finally answered. The purpose of this review is to begin to get a consensus as to what the most important of those remaining questions are. Some of the earliest observations of inflammatory defects associated with cancer were those of Berg, who in 1959 reported that there was a significant difference in inflammatory infiltrates in breast cancer biopsies in patients who were long-term sur-

Court,

Suite

200,

(USA)

vivors, compared with those who were not. In 1961 Mahoney and Leighton demonstrated that the inflammatory responses to implanted cotton thread were impaired in tumours of mice and rats compared with the responses which occurred in non-neoplastic tissue. Several years later, in 1963 Dizon and Southam demonstrated experimentally, using a Rebuck skin window technique, that breast cancer patients had defective macrophage migration activity. A number of studies on the potential role of macrophages in tumour growth and the ability of tumours to suppress macrophage function were initiated using animal models and reported in the early 1970s. Among these were some studies of Eccles and Alexander (1974), which demonstrated an inverse correlation between macrophage content and metastatic potential. Another study was that of Russell ef al. (1976), which showed that regressing

MACROPHAGES

Moloney sarcomas contained as many as five times the number of macrophages as did progressively growing sarcomas. Studies by Fauve et al. in 1974 marked one of the turning points in this field of research, as they were one of the first to demonstrate that antiinflammatory activity associated with neoplasms could be demonstrated with soluble factors released from such turnours. The concept that cancers might subvert the macrophage inflammatory response, and perhaps concurrently the anti-tumour response, by release of soluble effector molecules was a particularly attractive one. It meant that one might now identity a definitive point of therapeutic intervention. If one could antagonize the effector molecules, one could prevent the depressed inflammatory responses and the immune system could function normally. A number of studies from several different labs soon appeared, confirming that macrophages from tumour-bearing animals had depressed migratory responses or that extracts prepared from various tumour tissues, when injected into normal animals, had potent antiinflammatory activity. Among such studies were those of Snyderman and Pike (1976), Stevenson and Meltzer (1976) and Normann et al. (1979). Several years passed in which papers from numerous labs confirmed the ability to suppress macrophage responses in normal mice by injection into those mice of passaged tumour cells or extracts prepared from such tumours. It was in the late 197Os, when researchers in the field became aware that many of the tumours being used were infected with lactic dehydrogenase virus (LDH), that many of the antiinflammatory effects being attributed to tumours and their products could be reproduced with purified preparations of LDH. Indeed, in our own lab we found that many of our routinely passaged tumours were contaminated as such, forcing us to redo some of our studies with tumours demonstrated to be free of LDH. In 1979 Snyderman and Cianciolo confirmed that the antiinflammatory effect of transplanted tumour cells and tumour cell extracts could be demonstrated using materials completely free of LDH and that no LDH could be detected in recipient mice. However, many of the earlier reports of tumour-associated antiinflammatory activity obtained with transplanted tumours must obviously be viewed with caution, given the potential for LDH contamination. As the decade of the 1980s began, there was considerably less interest in the role of antiinflammatory products released from tumours. This was perhaps due to the “scare” associated with the potential of artifactual results due to LDH or to the fact that as the decade of molecular biology began to gather speed, it was advantageous to work with well-defined effector molecules that would rapidly lend themselves to molecular cloning and sequencing. Although we

AND

CANCER

269

(Snyderman and Cianciolo) had great confidence that a tumour-associated antiinflammatory factor(s) did indeed exist, the potency of this factor(s) had made it difficult to obtain the amounts and purity necessary for characterization. Physicochemical similarities to a recently identified type C retroviral transmembrane protein, P15E, suggested that this protein might be a candidate. We (Cianciolo et a/., 1980, 198 1, 1983, 1984a) subsequently demonstrated that P15E (free of any potential contamination with LDH) was itself a potent antiinflammatory agent when injected into normal mice and that both murine tumours as well as human malignant cells and fluids contained P 1SE-related antigenic materials. We subsequently reported (Cianciolo et al., 1984b) that a highly conserved region of P15E, representing 26 amino acids, was responsible for much of its biological activity and that this conserved region was found in a large number of viruses from a wide variety of species including murine, feline, bovine, avian, simian and even human. Over the last seven years a large number of studies have reported that peptides corresponding to this highly conserved region of P15E have a wide range of biological activities including, but not limited to, the suppression of a variety of macrophage responses. The issue which has not been resolved however, is the relationship of P 1SE-related proteins to the antiinflammatory activities of tumours. Nelson et al. (1985) demonstrated that the antiinflammatory effect of bovine tumour extracts injected into normal mice could be abrogated with monoclonal antibodies to P15E and that growth of transplanted murine tumours could be significantly delayed by in uivo treatment with such monoclonal anti-Pl5E antibodies. Recently, Lindvall and SjGgren (1991) reported in vivo inhibition of rat yolk sac tumours with antiP15E monoclonal antibody therapy. A number of recent studies have confirmed the earlier observation that human malignancies contain P 1SE-related antigens. Tan et al. (1986) demonstrated that tumours from virtually 100 070of patients with head and neck cancers contained PlSE-related antigens as demonstrated histochemically and that the inhibitory activity for monocyte chemotaxis found in the sera of such patients could be successfully removed with P15E antibodies. Van de Plassche-Boers et al. (1988) and Tas et al. (1991) reported studies which showed that PlSE-related inhibitors of monocyte chemotaxis were not found exclusively in malignancy but could also be found in such conditions as chronic purulent rhinosinusitus or Graves’ disease. Such studies support the concept that PlSE-related antiinflammatory factors associated with neoplasia may be proteins with a normal immunoregulatory role and these proteins are overexpressed in malignancy or chronic inflammation.

270

50th FORUM

Further support for such a concept comes from our recent observations that many of the biological activities described for P 15E and related proteins are similar to those described for transforming growth factor beta (TGF-P), an immunoregulatory protein overexpressed in a variety of malignant cells. We (Cianciolo, 1990) recently identified a region of partial homology between TGF-P and the region of P15E tentatively identified as its active site. A peptide corresponding to this region of TGF-P is able to mimic many of the biological activities of TGF-P. More importantly, perhaps, we have found (unpublished observation) that at least one monoclonal antibody to P15E is able to absorb radiolabelled TGF-P from solution. Whether some or all tumour-associated antiinflammatory activities are related to TGF-P remains to be determined. Recently, Lindeskog et al. (1993) have reported the isolation using polymerase chain reaction of human endogenous viral sequences corresponding to the immunosuppressive region of P15E. Previous studies from other groups have also indicated the presence of such homologous sequences, but these were not expressed because of stop codons. In summary, the studies begun nearly twenty years ago appear to have been confirmed in that a number of different labs have now reported antiinflammatory proteins associated with neoplasms of both rodent and human origin. It is unlikely that these activities are due to LDH or mycoplasma contamination as many of the studies are based on the specific removal of activity using one or more monoclonal antibodies. Indeed, R.A.J. Oostendorp (Amsterdam) has recently found (personal communication) that a monoclonal antibody prepared specifically to the immunosuppressive region of P 15E reacts histochemitally with a variety of human neoplasms and absorbs antiinflammatory activity from human tumour materials. However, the question still remains, particularly in the case of human neoplasia, as to the specific nature of these materials. Are they endogenous human retroviral sequences which are being turned on? Are they one of the known growth factors, such as TGF-P, or perhaps an as yet unidentified growth factor which is overexpressed in tumour tissues? Or are they perhaps completely unique proteins which have similarities to both of the above but represent their own class or family of immunoregulatory proteins? It is clear that the definitive answer to these questions will require that we use all of the technical tools which have become available over the last decade to isolate, sequence, clone and express these factors.

References Berg, J.W. (1959), Inflammation and prognosis in breast cancer. Cancer, 12, 714.

IN IMMUNOI~OGY Cianciolo, G.J., Matthews. T.J., Bolognesi, D.P. & Snyderman, R. (1980), Macrophage accumulation in mice is inhibited ived from murine

by low molecular leukemia viruses.

weight factors J. lmnunol.,

der-

124, 2900-2905. Cianciolo, G.J., Hunter, J., Silva, J., Haskill, J.S. & Snyderman, R. (1981), Inhibitors of monocyte responses to chemotaxis are present in human cancerous effusions and react with monoclonal antibodies to the Pl5E structural protein of retroviruses. J. Clin. Invesr., 68, 831-844. Cianciolo, G.J., Lostrom, M.E., Tam, M. & Snyderman, R. (1983), Murine malignant cells synthesize a 19,000-dalton protein physicochemically and antigenically related to the immunosuppressive retroviral protein P 15E. J. exp. Med., 158, 885-900. Cianciolo, G.J., Phipps, D. & Snyderman, R. (1984a), Human malignant and mitogen-transformed cells contain retroviral PlSE-related antigen. J. exp. Med., 159, 964-969. Cianciolo, G.J., Kipnis, R.J. & Snyderman, R. (1984b), Similarity between Pl5E of murine and feline leuke-

mia viruses and P21 of HTLV. Nalure (Lond.). 311, 515.

Cianciolo, G.J. (1990), Inhibition of lymphocyte proliferation by a synthetic peptide corresponding to a region of transforming growth factor beta homologous to CKS-17, an immunosuppressive retroviral-related peptide. Clin. Res., 38, 325A. Dizon, Q. & Southam, C.M. (1963), Abnormal cellular response to skin abrasion in cancer patients. Cancer, 16, 1288-1292. Eccles, S.A. & Alexander, P. (1974), Macrophage content

of tumors in relation to metastatic spread and host immune reaction. Nufure (Lond.), 250, 667-669. Fauve,

R.M.,

Hevin,

B.,

Jacob,

H.,

Gaillard,

J.A.

&

Jacob, F. (1974), Antiinflammatory effects of murine malignant cells. Proc. nut. Acad. Sci. (Wash.), 71, 4052. Lindeskog, M., Medstrand, P. & Blomberg, J. (1993), Sequence variation of human endogenous retrovirus ERV-9 related elements in an env region ing to an immunosuppressive peptide:

in normal 1122-l

and neoplastic

correspond-

transcription cells. J. Viral., 67,

126.

Lindvall, M. & Sjiigren, H.O. (1991), Inhibition of rat yolk sac tumor growth in vivo by a monoclonal antibody to the retroviral molecule P 15E. Cancer Immunol. fmmunorher., 33, 21-27. Mahoney, M.J. & Leighton, J. (1961), The inflammatory response

to

a foreign

body

within

transplantable

tumors. Cnncer Res., 22, 334-338. Nelson, M., Nelson, D.S., Spradbrow, P.B., Kuchroo, V.K., Jennings, P.A., Cianciolo, G. J. & Snyderman, R. (1985), Successful tumor immunotherapy: possible

role

of antibodies

to antiinflammatory

factors 61,

produced by neoplasms. Clin. exp. Immunol., 109-l

17.

Normann, S.J., Schardt, M. & Sorkin, E. (1979), Antiinflammatory effect of spontaneous lymphoma in SJL/J mice. J. nut. Cancer Inst., 63, 825-833. Russell, S.W., Doe, S.F. & Cochrane, C.G. (1976) Number of macrophages and distribution of mitotic activity in regressing and progressing Moloney sarcomas. J. Immunol., 116, 164-166.

MACROPHAGES Snyderman, R. & Cianciolo, G.J. (1979), Further studies of a macrophage chemotaxis inhibitor (MCI) produced by neoplasms: murine tumors free of lactic dehydrogenase virus produce MCI. J. Reficuloendofh. Sot., 26, 453-458. Snyderman, R. &Pike, M.C. (1976), An inhibitor of macrophage chemotaxis produced by neoplasms. Science, 192, 370-372. Stevenson, M.M. & Meltzer, M.S. (1976), Depressed chemotactic responses in vitro of peritoneal macrophages from tumor-bearing mice. J. nut. Cancer Insr., 57, 847-852. Tan, I.B., Drexhage, H.A., Scheper, R.J., von Blombergvan de Flier, B.M.E., de Haan-Meulman, M., Snow, G.B. & Balm, A.J.M. (1986), Immunosuppressive

AND

271

CANCER

retroviral P 1SE-related factors in head and neck carcinomas. Arch. Ololaryngol. Head and Neck Surg., 112, 942-945. Tas, M., de Haan-Meulman, M., Kabel, P.J. & Drexhage, H.A. (1991), Defects in monocyte polarization and dendritic cell clustering in patients with Graves’ disease. A putative role for a non-specific immunoregulatory factor related to retroviral P15E. C/in. Endocrinol., 34, 441-448. Van de Plassche-Boers, E.M., Tas, M., de Haan-Meulman. M., Kleingeld, M. & Drexhage, H.A. (1988), Abnormal monocyte chemotaxis in patients with chronic purulent rhinosinusitis : an effect of retroviral Pl5Erelated factors in serum. Clin. exp. Immunol., 73, 348-354.

The macrophage response to infectious agents: mechanisms of macrophage activation and tumour cell killing R. Keller Department

Pathologic der Universittit, Institut fiir Experimenrelle Stern watstrasse 2, CH-8091 Zurich (Switzerland)

Mononuclear phagocytes have long been considered major effecters of host defence against microbial, and in particular, bacterial, infection. In showing that any agent that stimulated T lymphocytes could also trigger bacteriostatic and bactericidal macrophage activities, George Mackaness was the first to provide evidence for the existence of a link between the T lymphocyte and macrophage activation (Mackaness, 1969). Later work identified interferon-y (IFNy) as the major macrophageactivating lymphokine. Macrophages that had been primed with this cytokine could be fully activated by lipopolysaccharide (LPS) (Rucco and Meltzer, 1978; Weinberg et a/., 1978; Meltzer, 1981). It is now generally agreed that stimulation by T cell-derived lymphokines represents a major pathway of paracrine macrophage activation. Another important development arose from episodic clinical observations that, in tumour patients, concomitant severe infectious diseases could result in a considerable decline in tumour growth. The experimental analysis of the clinical reports, in partic-

Immunologic,

ular by W.B. Coley, led to the identification of tumour necrosis factor by L.J. Old and his colleagues (Carswell et al., 1975) and established a link between mononuclear phagocytes and tumours. Utilizing pure lymphocyte-free bone-marrowderived mononuclear phagocytes (BMMP) from rodents and humans as the source for macrophages, we have analysed over the past few years the consequences of their interactions with microorganisms, in particular, bacteria and bacterial products (LPS, lipid A, peptidoglycan, lipoteichoic acid). In the course of this work, various points at issue arose, some of which will be briefly discussed here; these include : a) the existence and operation of an autocrine pathway of macrophage activation; b) the pattern of the macrophage defined bacterial structure; and

response to a

c) the mechanisms involved in tumour cell killing by activated macrophages.