Immunoregulatory role of in vitro differentiated macrophages on human natural killer (NK)-cell activity

Immunoregulatory effects of human macrophages on natural killer (NK) activity were studied. Monocytes were isolated by adherence to plastic, after leukapheresis of normal blood donors, and cultured for 1 to 14 days. In vitro-differentiated (5-7 days) human macrophages consistently and significantly (P less than 0.01) augmented NK activity of fresh autologous or allogeneic PBMNC. During culture, these macrophages also developed increased antitumor cytostatic activity. The optimal time for both the expression of cytostatic activity and up-regulation of NK activity was 5-7 days in culture. In contrast, 12- to 14-day macrophages significantly suppressed NK activity and had less cytostatic activity. Macrophages in culture demonstrated shifts in Leu-M3+HLA-DR+ phenotype from the mean of 60% +/- 11 (SD) in fresh monocytes to 90% +/- 5 between Days 5 and 7 in culture and then down to 10% +/- 5 in 14-day cultures. The activity of NK (CD56+CD3-) cells, purified by Percoll gradient centrifugation and flow cytometry, was up-regulated directly by in vitro-differentiated macrophages at low macrophage to NK cell ratios, and this up-regulation was not dependent on T lymphocytes or other accessory cells. The modulation of NK activity by differentiated macrophages was not MHC-restricted and depended on the viability and cellular integrity of macrophages. Sonicated macrophages could no longer up-regulate NK activity. This study shows that antitumor effects mediated by human in vitro differentiated LeuM3+HLA-DR+ macrophages may simultaneously involve more than one mechanism, namely direct cytostasis of tumor cells and activation of NK cells.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses. I. Tumor-specific Lyt-1+2- T cells are required for producing MAF able to generate cytolytic as well as cytostatic macrophages

In the present study we investigated some of the cellular mechanisms for the generation of macrophage-activating factor(s) (MAF) in immune responses to tumor antigens. C3H/HeN mice were immunized to syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma by intradermal inoculation of viable tumor cells, followed by the surgical resection of the tumor. Spleen and lymph node cells from these tumor-immune mice were stimulated in vitro with the corresponding tumor cells, and supernatant from such a culture was tested for an ability to activate macrophages to exert their cytostatic and cytolytic activities as detected on tumor cells unrelated to immunizing tumors. Peritoneal adherent cells as a macrophage source, which were preincubated with supernatant from co-culture of tumor-unimmunized normal spleen and lymph node cells plus tumor cells, failed to exhibit any significant antitumor effect on unrelated X5563 tumor cells, whereas the addition of supernatant from cultures containing immune lymphocytes to adherent cells resulted in appreciably potent cytostatic and cytolytic effects on X5563 tumor cells, indicating the generation of MAF in culture supernatant. The activation of tumor-immune spleen and lymph node cells for MAF generation was tumor-specific, because anti-MH134- and anti-MCH-1-A1-immune lymphocytes produced MAF by the stimulation with the respective but not with the other alternative tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 but not with anti-Lyt-2.1 antibody plus complement before culturing. These results indicate that the tumor-specific Lyt-1+2- T cell subset has a crucial role in generating MAF by which an adherent cell population as a source of macrophages acquires the potential for inducing a cytolytic as well as a cytostatic effect on tumor cells.     

Effect of recombinant IFN-gamma (rIFN-gamma) on the mechanism of human macrophage IgG FcRI-mediated cytotoxicity. rIFN-gamma decreases inhibition by cytophilic human IgG and changes the cytolytic mechanism.

Different classes of receptors for the Fc moiety of IgG (Fc gamma R) have been defined on human monocytes and macrophages: Fc gamma RI, Fc gamma RII, and Fc gamma RIII. All three classes are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Fc gamma RI, which binds monomeric human IgG (hIgG) with high affinity, was shown an effective cytotoxic trigger molecule on different types of cells. In vitro, the inhibition of Fc gamma RI-mediated ADCC by hIgG is well documented. The low affinity receptor classes, Fc gamma RII and Fc gamma RIII, are not blocked by monomeric hIgG. Because monomeric hIgG is present at high concentrations in plasma and interstitial fluids it has been postulated inhibitory in vivo. We investigated the effect of rIFN-gamma on macrophage Fc gamma RI-mediated ADCC in the presence of low doses hIgG. With human E sensitized with hIgG as target cells, Fc gamma RI was studied selectively. We found that rIFN-gamma enhances both expression and cell surface density of Fc gamma RI on cultured peripheral blood monocytes. Furthermore, this cytokine partially reversed the inhibitory effect of monomeric hIgG on ADCC. More interestingly, we found that the cytolytic mechanism of monocyte-derived macrophages changed completely after prolonged culture with rIFN-gamma. Monocytes cultured for 9 days in control medium mediate predominantly phagocytosis. After long term rIFN-gamma stimulation (9 days), monocyte-derived macrophages almost completely lost the capacity to perform phagocytosis. Interestingly, they became highly efficient in mediating extracellular lysis of human E sensitized with hIgG. Short term rIFN-gamma stimulated monocyte-derived macrophages (for the last 40 h of culture) were found to mediate both phagocytosis and extracellular lysis. Our findings suggest that in vivo rIFN-gamma-stimulated macrophages may be most efficient in Fc gamma RI-mediated cytolysis as a consequence of a changed cytolytic mechanism in combination with enhanced Fc gamma RI density.     

Rejection of murine ovarian cancer following treatment with regional immunotherapy: correlations with a neutrophil-mediated activation of cytostatic macrophages

Rejection of the murine ovarian teratocarcinoma (MOT) in C3HeB/FeJ mice, following intraperitoneal (ip) treatment with Corynebacterium parvum (C. parvum), is abrogated by injections of silica. We, therefore, investigated whether C. parvum-elicited macrophages affect MOT targets in vitro. Tumor-cytostatic, but not cytolytic, macrophages were detected in normal and tumor-challenged mice treated with C. parvum. The dose responsiveness and kinetics of macrophage activation strongly correlated with tumor rejection. A pyridine extract of C. parvum, possessing greatly diminished tumor rejection properties, was significantly less effective in activating macrophages. Cytostatic macrophage activation and prevention of tumor outgrowth also followed treatment in C3H/HEJ mice, a strain with a known deficiency in cytolytic macrophage function. Peritoneal neutrophils, obtained 6 hr after treatment with C. parvum, were capable of activating cytostatic macrophages when reinjected ip into normal mice. These results indicate a critical role for tumor cytostatic macrophages in this immunotherapy model and suggest their activation is mediated by inflammatory neutrophils.     

Evidence for cytostatic T cell activity in the effector mechanism against syngeneic TMT mammary tumor cells in mice

The effector mechanism of immune spleen cells against syngeneic TMT mammary tumor cells was analyzed in vitro. C3H/He mice were first inoculated with TMT tumor cells, and then the tumors were x-irradiated with 2000 rad 1 wk after the inoculation. Spleen cells from these treated mice inhibited the growth of tumor cells in vitro when assessed by (3H)-TdR incorporation by tumor cells (cytostatic activity). The same spleen cells did not have any cytotoxic activity on TMT tumor cells detected by a 51Cr-release assay. The cytostatic activity was mediated by Lyt-1+23- T cells. The purified T cells alone could not inhibit the growth of tumor cells, but accessory cells were required for the induction of cytostatic T cell activity. The accessory cells were Ia-positive, macrophage-like adherent cells. Furthermore, both T cells and macrophages were also required for the inhibition of tumor growth even after the spleen cells were activated in vitro. These results suggest T cells and macrophages play an important role in the effector mechanism against TMT mammary tumor cells. The mechanism of cytostasis by T cells and macrophages was discussed from the standpoint of the cellular interaction.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.     

Interactions between human macrophages and tumor cells in three-dimensional cultures

Human blood mononuclear cells were cultured for 7 days in hydrophobic plastic bags. Macrophages differentiated from monocytes and purified by elutriation were then cocultured with round-shaped aggregates of epithelial cells (spheroids). Spheroids prepared from the SK-MES-1 carcinoma cell line were cultured individually, under constant stirring, in multiwell plates coated with agarose. Macrophage/spheroid interactions were investigated under various experimental conditions. Macrophages activated with interferon aggregated to each other and to spheroids, in contrast to control unactivated macrophages. Histological examination, after staining with a macrophage-specific monoclonal antibody, showed that both control and interferon--activated macrophages migrated between epithelial tumor cells and infiltrated the spheroids. The addition of anti-ICAM-1 monoclonal antibody inhibited macrophage homotypic aggregation as well as aggregation to and penetration into spheroids. The macrophages did not exert cytolytic effects, as judged by a chronium-51 release assay, but provoked a diminution of tritiated thymidine incorporation by tumor cells. Cytostatic activity was observed with effector: target ratios as low as 116, and was maximal (99% at a 11 ET ratio) with macrophages differentiated in the presence of granulocyte/macrophage-colony-stimulating factor. The cytostatic effect was not related to tumor necrosis factor secretion.Work supported by Association pour la recherche sur le cancer (ARD)     

Synergistic effect of concanavalin A and Bu-WSA on DNA synthesis in human peripheral blood lymphocytes

Butanol-extracted water soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was not mitogenic for human peripheral blood mononuclear cells (PBM) but was capable of enhancing (3H) thymidine uptake of T cells stimulated by concanavalin A (Con A) in the presence of B cells or macrophages (M phi) in vitro. The mechanisms of the synergy of Con A and Bu-WSA were studied by using separated cell populations from PBM. Both subfractioned OKT4+ and OKT8+ cells were responsive to co-stimulation by Con A and Bu-WSA in the presence of an accessory cell population. Allogeneic B cells and M phi as well as autologous cells had helper function as accessory cells. Heavy irradiation with gamma-rays did not affect the function of the accessory cells, but previous treatment of B cells with anti-Ig serum plus complement (C) or treatment of M phi with anti-M phi serum plus C deprived them of their function. The treatment of accessory cells with anti-HLA-DR serum, regardless of the presence or absence of C, resulted in loss of their helper function. Cultures in Marbrook-type vessels showed that a mixed cell population of T cells and accessory cells in the lower chamber produced some active factor(s) after co-stimulation with Con A and Bu-WSA, and by passing through the membrane filter separating the chambers, the factor(s) enhanced the proliferation of the Con A-activated T cell population in the upper chamber. The factor(s) was presumed to be interleukin 2 (IL 2), because it supported the growth of IL 2-dependent CTLL cells. These results indicate that the synergy of Con A and Bu-WSA on the proliferative response of human PBM is due to the elevation of growth factor production from T cells stimulated by those mitogens.     

Expression of the human papillomavirus E7 oncogene during cell transformation is sufficient to induce susceptibility to lysis by activated macrophages.

Human papillomaviruses (HPV), and in particular HPV type 16, are etiologic agents in the development of cervical cancer, which is the second most common form of cancer in women worldwide. Mammalian cells are susceptible to transformation in vitro by the E6 and E7 oncogenes derived from the HPV-16 genome. NIH-3T3 cells transfected with the HPV-16 E7 oncogene were found to exhibit cytolytic susceptibility to murine-activated macrophages. In comparison, E6 oncogene-expressing cells were not susceptible to lysis by activated macrophages. The E7 oncoprotein is multifunctional, being capable of complexing with the retinoblastoma tumor suppressor gene (anti-oncogene) product, stimulating DNA synthesis, and causing cell transformation in vitro. Macrophage killing assays performed on cell lines expressing E7 mutants revealed that the ability to complex the retinoblastoma tumor suppressor gene product and stimulate DNA synthesis did not induce susceptibility to activated macrophages, whereas the ability of E7 to cause transformation was required to induce susceptibility to activated macrophages. These data suggest that cell transformation is a more important prerequisite for inducing susceptibility to activated macrophages than is the loss of tumor suppressor gene function. This study also provides an initial link between HPV-16 oncogene expression and the ability of activated macrophages to selectively recognize and destroy HPV-16-associated neoplastic cells.     

Growth inhibitory factor of human alveolar macrophage

The effect of human lung conditioned medium (HLCM) on the DNA synthesis in cultured human alveolar macrophages (HAM) was evaluated. The medium from human lung cultured for 3 days (3d-HLCM) promoted the DNA synthesis as well as the recombinant human GM-CSF does. On the other hand, that cultured for six days (6d-HLCM) did not promote the DNA synthesis but strongly suppressed GM-CSF induced DNA synthesis in HAM. This growth inhibitory effect was also observed when several macrophage like cell lines were cultured with 6d-HLCM. Partial purification of an inhibitory factor on gel permeation HPLC revealed two distinct peaks of activity with molecular weights of 38 kd and 110 kd.     

Cytolytic response of human T cells against allogeneic small cell lung carcinoma treated with interferon gamma

Summary Human lymphocytes stimulated in vitro by allogeneic small cell lung carcinoma cell lines did not show any significant cytolytic activity against the stimulator tumor cells. However, a high level of lysis was observed when both stimulator and target small cell lung carcinoma cells were pretreated with inferferon , which increased considerably the expression of major histocompatibility class I molecules by these cells. The demonstration that small cell lung carcinoma cells can be lysed by cytolytic T lymphocytes, suggests that it will be feasible to study the autologous T cell response of patients against this tumor.     

In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients

Peripheral blood monocytes (PBM) from AIDS patients have exhibited defects in some but not all of the immune functions yet tested. This study has examined the capacity of AIDS PBM to lyse tumor target cells as well as their ability to secrete TNF. Untreated PBM from AIDS patients were significantly cytotoxic to U937 target cells and responded to IFN-gamma pretreatment with augmented cytotoxicity. Both the spontaneous and IFN-gamma-stimulated cytotoxic activity was significantly (p less than 0.01) higher than that observed with normal PBM. The cytotoxic activity depended on the E:T ratio used and was higher in AIDS PBM at all ratios tested (10:1 to 40:1). Because TNF has been implicated in macrophage cell-mediated cytotoxicity, we examined whether the elevated cytotoxic activity of AIDS PBM was associated with an increase in TNF production. Supernatants from PBM cultured overnight with or without IFN-gamma were tested in a bioassay measuring cytotoxicity against U937 target cells as well as in an RIA specific for TNF. Supernatants derived from either unstimulated or IFN-gamma-treated AIDS PBM exhibited significantly higher levels of cytotoxicity than supernatants from normal macrophages. Both normal and AIDS PBM produced higher levels of cytotoxic factors in response to IFN-gamma. As determined by the RIA, AIDS PBM spontaneously released high levels of TNF whereas little TNF was produced by normal PBM. Treatment with IFN-gamma augmented the level of TNF production in both AIDS and normal PBM. These results demonstrate that PBM from AIDS patients have undergone in vivo activation as manifested by both cytotoxicity against tumor target cells and production of TNF. Target cell lysis by both AIDS PBM and their supernatants was inhibited by monoclonal anti-rTNF, suggesting that the increase in PBM cell-mediated cytotoxicity was caused by an increase in TNF production. The significance of these findings in the pathogenesis of the disease is discussed.     

The Interaction of Acanthamoeba spp. with Activated Macrophages and with Macrophage Cell Lines

Acanthamoeba spp. are free-living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B₆C₃F₁ mice after intranasal challenge with Acanthamoeba-induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with ³H-uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro-mium-51 from radiolabeled macrophages. Acanthamoeba culbensoni , which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba -mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba -specific antibodies increased the amebicidal activity of activated macrophages. Macrophage-mediated destruction was by contact-dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.     

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.     

Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice: augmentation of anti-tumor cytostatic activity

A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DFR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effectors, respectively. The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. However, a sufficient number of macrophages were always required as the final effectors in their functional expression. These primary effectors were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seems to exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responsible for cytostatic anti-tumor immunity.     

Parameters affecting the in vitro maturation of human monocytes to macrophages

In vitro maturation of human monocytes to macrophages was characterized by morphological criteria, cell size and lysosomal enzymes activity. Purified populations of monocytes were maintained in culture at either adherent or nonadherent conditions and their maturation to macrophages was observed in both cases. The addition of external factors such as hydrocortisone and vitamin D3 inhibited monocyte maturation. In the absence of external factors, nonadherent monocytes were inhibited in their maturation for up to 10 days when plated at crowded cell concentrations. In addition, the presence of human serum in the culture media had a higher inhibitory activity than similar concentrations of fetal calf serum. Supernates from crowded macrophages were also inhibitory for monocyte maturation. We suggest the possibility that cell crowding, as well as soluble factors found in the serum and probably secreted by macrophages, participate in the regulation of monocyte development by inhibiting their maturation. Once released from this inhibitory signal or environment, the monocytes mature to macrophages.     

Modulation of collagen production following bleomycin-induced pulmonary fibrosis in hamsters. Presence of a factor in lung that increases fibroblast prostaglandin E2 and cAMP and suppresses fibroblast proliferation and collagen production

To elucidate mechanisms involved in the regulation of lung collagen content we studied hamsters with bleomycin-induced pulmonary fibrosis. Lung collagen in this model is increased as the result of greatly increased lung collagen synthesis rates. However, collagen synthesis rates are subsequently restored to normal. Hamster lung explants from both normal and bleomycin-exposed hamsters were cultured, and the effects of explant conditioned medium (CM) on lung fibroblast (IMR-90) proliferation and collagen production in vitro were determined. Lung explant CM increased fibroblast prostaglandin (PG)E2 production and intracellular cAMP, and decreased both fibroblast proliferation and collagen production in a dose-dependent manner. Greater activity was observed with lung explant CM from bleomycin-exposed lungs. Incubation of fibroblasts with indomethacin prior to addition of CM blocked CM-mediated changes in PGE2 and cAMP and inhibited changes in fibroblast proliferation and collagen production. Exogenous PGE2 or dibutyryl cAMP also suppressed fibroblast proliferation and collagen production. The suppressive activity in lung-conditioned medium is nondialyzable, has an apparent molecular weight of 15,000-20,000 by gel filtration, and is heat-stable. It is not species-restricted since CM from hamster lung affected human and hamster lung fibroblasts similarly. Activity is present preformed in lung and bronchoalveolar lavage fluid, although bronchoalveolar macrophages produce a nondialyzable factor in culture which suppresses fibroblast proliferation. The suppressive activity identified in fibrotic lung may represent a means for limiting collagen accumulation following tumor injury.     

Spontaneous cytotoxicity of macrophages against pancreatic islet cells

Activated peritoneal macrophages were found to lyse syngeneic [3H]leucine-labeled pancreatic islet cells or rat insulinoma cells after 15 h of coculture at 37 degrees C. Lysis was verified by electron microscopic analysis. Islet cell lysis was dependent on the T:E ratio and was comparable with P815 and L929 tumor cells used as targets. The cytotoxic activity was localized in the adherent fraction of Corynebacterium parvum activated peritoneal cells and was destroyed by incubation of cells with macrophage-toxic silica particles. Syngeneic thyrocytes and hepatocytes were found to be resistant to the cytolytic action of activated macrophages. It has been shown previously that macrophages contribute to pancreatic islet inflammation. The present in vitro analysis demonstrates that macrophages can function as effector cells in islet destruction.     

Comparison of the cytostatic and cytolytic activity of tumor necrosis factor-alpha and interleukin 1 alpha in human malignant cell lines.

The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 alpha (IL-1 alpha) share many properties, including in-vitro cytotoxicity. Because cytotoxicity can result from either cytolytic or cytostatic activity, and because differentiating between these activities may have clinical relevance, we determined the cytostatic and cytolytic activity of TNF-alpha and IL-1 alpha for the human cell lines ME-180, SiHa (cervical carcinomas) and A375 (melanoma). Results of these analyses showed that IL-1 alpha mediated cytostatic activity only for A375 cells. IL-1 alpha was not cytolytic in the presence or absence of protein synthesis inhibitors. TNF-alpha was cytostatic for A375 and ME-180 cells, and although TNF-alpha was not cytolytic in the absence of protein synthesis inhibitors, it was cytolytic in the presence of protein synthesis inhibitors. These results suggest that the difference between the cytolytic and cytostatic activities of IL-1 alpha and TNF-alpha may have therapeutic implications for the use of these biological response modifiers in the treatment of gynecological malignancies.