Tumoricidal activity of alveolar and peritoneal macrophages of C57BL/6 mice bearing metastatic or nonmetastatic variants of Lewis lung carcinoma.

The spontaneous tumoricidal abilities of alveolar and peritoneal macrophages from C57B1/6 mice bearing a metastatic or a nonmetastatic cloned variant of Lewis lung carcinoma (LLC) were measured. Cytotoxicity by alveolar macrophages was enhanced during the first few weeks after subcutaneous (s.c.) or intravenous (i.v.) injection of metastatic LLC-C3 cells, but not after injection of nonmetastatic LLC-C8 cells. Alveolar macrophages from mice with s.c.-injected metastatic tumors, but not with nonmetastatic tumors, could be further activated in vitro, but not beyond the maximal level of spontaneous cytotoxicity. Late in tumor growth, the spontaneous cytotoxicity by alveolar macrophages of metastatic LLC-C3 tumor bearers was suppressed and could not be increased by in vitro activation. The tumoricidal abilities of peritoneal macrophages from mice bearing either LLC-C3 or LLC-C8 tumors were modulated in a similar way, as were alveolar macrophages. The reduced cytotoxicity by alveolar macrophages from mice with nonmetastatic tumors or from mice bearing large metastatic tumors was not due to suppression by macrophage-derived prostaglandins. The loss of tumoricidal capabilities by macrophages from mice with large metastatic LLC-C3 tumors was not caused by elevated systemic prostaglandin E2 (PGE2) levels. These results suggest that alveolar and peritoneal macrophages are activated to be cytotoxic during development of pulmonary metastases and do not need to be functionally depressed for successful establishment of metastases.

Association of a functional prostaglandin E2-protein kinase A coupling with responsiveness of metastatic Lewis lung carcinoma variants to prostaglandin E2 and to prostaglandin E2-producing nonmetastatic Lewis lung carcinoma variants.

Cloned Lewis lung carcinoma (LLC) variants were used in an in vitro migration model for dissemination, to determine if prostaglandin E2 (PGE2) produced by nonmetastatic LLC cells could directly stimulate dissemination of metastatic LLC cells and to identify an intracellular mechanism for such an effect. The migration of metastatic LLC clones was stimulated not only by exogenous PGE2 but also by nonmetastatic LLC cells, by their production of a migration-stimulatory factor which was sensitive to indomethacin and anti-PGE2 antibodies. Nonmetastatic LLC clones were unresponsive to migration stimulation by PGE2. The results of in vivo metastasis studies were consistent with those of in vitro migration studies. In vivo lung metastasis was increased by PGE2, as well as by nonmetastatic cells when they were either admixed with the metastatic LLC inoculum, irradiated and injected adjacent to the metastatic LLC tumor, or localized in chambers and implanted s.c. into mice given injections of metastatic LLC cells. Indomethacin blocked metastasis stimulation by nonmetastatic cells. The in vitro PGE2 stimulation of metastatic LLC cells appeared to be linked to a cyclic AMP (cAMP) response, since migration could also be stimulated by dibutyryl-cyclic AMP and blockage of a cAMP response with nicotinic acid ablated the PGE2 stimulation of migration. In vivo metastasis could be stimulated by elevation of cAMP with aminophylline. The differential responsiveness of metastatic versus nonmetastatic LLC cells to PGE2 could not be due to PGE2-adenylate cyclase coupling, since PGE2 increased the cAMP levels in cultures of both metastatic and nonmetastatic LLC cells. There was, however, a difference in the cyclic AMP-dependent protein kinase (PKA) response to PGE2, with PKA activity of metastatic LLC being stimulated by PGE2 and by the adenylate cyclase-stimulator forskolin, whereas PKA of nonmetastatic LLC was not stimulated by these cAMP elevators, suggesting a dysfunction in the cAMP-PKA coupling.

The effects of 16,16-dimethyl PGE2 and phosphodiesterase inhibitors on Con A blastogenic responses and NK cytotoxic activity of mouse spleen cells.

Prostaglandins function as down regulators of immune responses probably by increasing the concentration of intracellular cAMP. Phosphodiesterase inhibitors, which prevent the breakdown of cAMP, also increase the intracellular levels of cAMP. Prostaglandins and phosphodiesterase inhibitors have both been shown to suppress immune responses in vitro. In this study 16,16-dimethyl PGE2 (dm-PGE2), added in vitro, suppressed the mouse spleen cell concanavalin A (Con A) response by 38% and natural killer (NK) activity by 53%. Addition of the phosphodiesterase inhibitors, theophylline, RO20-1724, or dipyridamole, decreased both the Con A response and NK activity by at least an additional 30%. We also demonstrate that treatment with dm-PGE2 and theophylline in vivo is more effective than either compound alone in inhibiting NK activity of both untreated mice and mice treated with polyinosinic-polycytidylic acid. These studies support the hypothesis that the immunosuppressive effect of dm-PGE2 is mediated by cAMP and suggest that treatment with a combination of dm-PGE2 and phosphodiesterase inhibitors can augment this immunosuppressive effect.

The effects of protein kinase inhibitors on lymphocyte blastogenesis and GVHD-induced splenomegaly.

The activation of the protein kinase C (PKC) pathway plays an integral part in the proliferation of many cell types including lymphocytes. We report that the PKC inhibitor H-7 caused inhibition of three commonly studied blastogenic responses (Con A, LPS, and MLR) with the strongest suppression being detected in the MLR. In contrast, HA1004, a potent inhibitor of cyclic nucleotide-dependent protein kinases, did not alter the blastogenic response but occasionally caused augmentation. The phenothiazine compounds studied inhibited the Con A and, to a lesser extent, the LPS responses. One of the compounds, promethazine-HCl, was effective in vivo in inhibiting splenomegaly resulting from the induction of graft vs. host disease. Our studies support the involvement of PKC in lymphoid blastogenesis. They also suggest that agents that can inhibit PKC activity may be useful in inducing immunosuppression in vivo.

The effects of E series prostaglandins on blastogenic responses in vitro and graft vs. host responses in vivo.

In this study the ability of prostaglandin E1 (PGE1), Misoprostol (a stable analog of PGE1), and 16,16-dimethyl PGE2 (a stable analog of PGE2) to suppress immune responses in vitro and in vivo was determined. All of the compounds caused a titratable (10(-6) to 10(-9) M) suppression of Con A blastogenesis and the mixed lymphocyte response whereas there was only slight inhibition of the LPS response. When either 16,16-dimethyl PGE2 (30 ug/mouse) or Misoprostol (60 ug/mouse) was administered daily in vivo, there was a significant suppression of splenomegaly in F1 mice (C57Bl/6 x CBA) which had been injected with parental (C57Bl/6) spleen cells. We conclude that prostaglandins of the E series can function as immunosuppressive reagents both in vitro and in vivo. In the future they may serve to augment existing forms of immunosuppressive therapy.

Suppressor alveolar macrophages in mice bearing metastatic Lewis lung carcinoma tumors.

The capacity of alveolar macrophages from mice injected with a metastatic Lewis lung carcinoma variant, LLC-C3, to regulate T-cell Con A blastogenesis and NK cytotoxicity was studied. During the first 5 days after subcutaneous tumor injection, alveolar macrophages were stimulatory to Con A blastogenesis of normal spleen cells. After 5 days, the alveolar macrophages shifted to become suppressive. The suppressive activity was extensive by day 11, when the primary and metastatic tumor foci were first detectable. The tumor-bearer alveolar macrophages also suppressed NK cytotoxicity. Alveolar macrophage suppressive activity was sensitive to indomethacin, suggesting a prostaglandin-dependent suppressor mechanism. Suppression was not mediated by the production of hydrogen peroxide or superoxide, as it was insensitive to catalase or superoxide dismutase. When normal alveolar macrophages were cultured with LLC-C3 supernatants for over 12 hours, suppressive activity was induced. The results of these studies show that alveolar macrophages of tumor bearers become suppressive with progressive tumor growth and might, thus, facilitate the development of pulmonary metastases.

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice.

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.

Adhesiveness and morphology of Lewis lung carcinoma variants influence their sensitivity to natural killer cytolysis and their metastatic capacity.

Cloned metastatic Lewis lung carcinoma cells, C3, were more resistant to natural killer (NK) lysis than were nonmetastatic variant cells, C8. This was influenced by the tumor cell adhesiveness and morphology. When the nonadherent round C3 cells were cultured with dimethylsulfoxide, they became adherent and spread, sensitive to NK lysis and less metastatic. When the adherent and spread C8 cells were made nonadherent and round with cytochalasin B, they became more resistant to NK lysis and more metastatic. These metastatic differences were not observed in 3-week-old NK-deficient mice.