Role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. II. Down-regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-macrophage colony-stimulating factor

Peritoneal elicited macrophages (PEM) from mammary tumor-bearing mice have a decreased capacity to become cytotoxic against syngeneic, allogeneic, and xenogeneic target cells upon in vitro stimulation with LPS, as compared with PEM of normal mice. A regulatory mechanism other than PG release is suggested because the addition of both indomethacin and LPS to macrophage cultures from tumor-bearing mice caused no changes in their cytotoxic capability. Because tumor products have been implicated in the down-regulation of immune responses, we investigated whether pretreatment with supernatants from the tumor cell line DA-3, derived from the in vivo mammary adenocarcinoma D1-DMBA-3, affects the cytolytic capacity of macrophages. This treatment inhibits, in a dose-dependent fashion, the ability of stimulated normal PEM to kill target cells. Partial purification of DA-3 cell line supernatant showed that most of the inhibitory activity was exerted by factors with a molecular mass greater than 10 kDa and less than 30 kDa. However, slight inhibition could also be observed with fractions containing molecules less than 10 kDa. The data suggest that more than one factor released by the mammary tumor cells may be involved in the down-regulation of macrophage-mediated cytotoxicity. Because the DA-3 cells constitutively produce granulocyte-macrophage CSF (GM-CSF), which has a molecular mass of 27 kDa, we pretreated PEM from normal mice in vitro with rGM-CSF for 24 h. This resulted in a dose-dependent decrease in their capacity to kill tumor target cells upon LPS stimulation. Furthermore, PEM from normal mice injected with rGM-CSF for 25 days displayed a profound decrease in their cytolytic ability against DA-3 targets upon in vitro stimulation with increasing amounts of LPS. The pretreatment of PEM from normal mice with a combination of DA-3 cell supernatants and specific anti-GM-CSF partially neutralized the inhibitory effect of the DA-3 supernatant on macrophage tumoricidal capability. These results indicate that tumor-derived GM-CSF is an important factor involved in the decreased macrophage cytotoxicity during mammary adenocarcinoma progression.     

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor

Summary Nonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against MM48 syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2^– and asialo-GM1⁺ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages. Abbreviations used: M-CSF, macrophage-colony-stimulating factor; NABMC, nonadherent bone marrow cells; CM, conditioned medium; NK, natural killer; N-CWS, Nocardia rubra cell-wall skeleton     

Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene

We have used transgenic mice that carry an activated c-neu oncogene driven by a mouse mammary tumor virus (MMTV) promoter to assess the stepwise progression of carcinogenesis in mammary epithelium. Unlike the stochastic occurrence of solitary mammary tumors in transgenic mice bearing the MMTV/c-myc or the MMTV/v-Ha-ras oncogenes, transgenic mice uniformly expressing the MMTV/c-neu gene develop mammary adenocarcinomas that involve the entire epithelium in each gland. Because these tumors arise synchronously and are polyclonal in origin, expression of the activated c-neu oncogene appears to be sufficient to induce malignant transformation in this tissue in a single step. In contrast, expression of the c-neu transgene in the parotid gland or epididymis leads to benign, bilateral epithelial hypertrophy and hyperplasia which does not progress to full malignant transformation during the observation period. These results indicate that the combination of activated oncogene and tissue context are major determinants of malignant progression and that expression of the activated form of c-neu in the mammary epithelium has particularly deleterious consequences.     

The regulatory effects of cytokines on the induction of a peripheral immunologic tolerance in mice.

Previous reports have demonstrated that injection of rIL-1 alpha into mice abrogates the ability of deaggregated human gamma-globulin (HGG) to induce a state of antigen specific immunologic tolerance in vivo. Our results demonstrate that human rIL-1 beta and a bioactive nonapeptide of human IL-1 beta inhibit the induction of tolerance to HGG suggesting that IL-1 affects tolerance induction through a noninflammatory mechanism of action because the immunoactive nonapeptide possesses only immunomodulatory properties. Further, TNF-alpha but not IL-6, cytokines with many bioactivities in common with IL-1, was found to inhibit the induction of tolerance. Therefore, it appears unlikely that IL-6 plays a role in the pathway by which either IL-1 or TNF-alpha interferes with tolerance induction. Although IL-2, IL-4, and IFN-gamma were incapable of directly affecting the induction of tolerance to HGG, it was determined that IL-4 and IFN-gamma were capable of inhibiting the ability of IL-1 to abrogate tolerance induction. It has been suggested that IL-1 induces the generation of endogenous IL-1 in vivo. Further, it has been demonstrated that IFN-gamma as well as IL-4 inhibits the synthesis of IL-1. Inasmuch as IL-4 and IFN-gamma inhibit the ability of IL-1 to abrogate tolerance induction, it appears that it is the endogenously generated IL-1 that interferes with tolerance induction. It was also determined that neither IL-4 nor IFN-gamma inhibits the activity of IL-1 which is consistent with results reported by others. Thus, results presented here suggest that the inhibition of tolerance induction to HGG by IL-1 may involve the stimulation of endogenous IL-1 synthesis.     

IgE-selective and antigen-specific unresponsiveness in mice. I. Induction of the unresponsiveness by administration of ovalbumin-pullulan conjugate.

An experimental model was demonstrated in mice for the induction of IgE-selective unresponsiveness to ovalbumin, a protein antigen. An administration of ovalbumin, conjugated with pullulan, a linear polymer of glucose, (OA-pullulan) into mice resulted in the induction of a long lasting, IgE-selective unresponsiveness to the subsequent immunization with native OA in the form optimal to elicit IgE antibody response. The IgE-selective unresponsiveness is antigen specific and is infectious to normal mice by transferring the spleen cells from mice receiving OA-pullulan conjugate at least 2 weeks before. In contrast to other modified antigens, OA-pullulan was found to elicit good IgM and IgG antibody responses, but not an IgE response, without the aid of an adjuvant.     

The enhancement of the immune response by pain stimulation in mice. I. The enhancement effect on PFC production via sympathetic nervous system in vivo and in vitro

Effects of catecholamines and osmotical and physical stimuli on the induction of anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) were investigated in (C57BL/6 X BALB/c)F1 mice in vivo and in vitro. The anti-SRBC PFC from mice immunized with 5 X 10(7) SRBC was markedly increased by daily s.c. injections of epinephrine. The enhancement of PFC by epinephrine was completely blocked by preadministration with propranolol and hexamethonium, but not with phentolamine. The PFC was increased by osmotic and physical stimuli given once a day for 4 days after immunization with SRBC. The enhancement of PFC by these stimuli was completely blocked by preadministration with propranolol and hexamethonium. The enhancement of PFC by physical stimuli was observed in nonimmunized mice when spleen cells from stimulated mice were cultured with SRBC in vitro. In normal mice, the enhancement of PFC was observed 2 hr after one physical stimulation. However, spleen cells from mice given two physical stimuli did not show the enhancement of PFC after treatment with anti-Thy-1.2 antibody and complement, nor after removal of nonadherent cells. Next, the serum obtained from mice 30 to 60 min after a physical stimulation enhanced PFC of normal mice spleen cells in vitro, but the enhancement was abolished by the addition of propranolol. The enhancement of anti-SRBC PFC by s.c. injection of epinephrine suggested that the autonomic nervous system, especially the sympathetic nervous system, was activated by a local stimulus effect of the injection. This enhancement of anti-SRBC PFC appear to be due to the activation of antigen non-specific helper T lymphocytes by the beta-actin of endogenous catecholamines from the adrenal gland.     

Induction of haem oxygenase contributes to the synthesis of pro-inflammatory cytokines in re-oxygenated rat macrophages: role of cGMP.

Macrophage activation and the resulting inflammatory response may be a major component of tissue injury upon hypoxia and re-oxygenation. Activation of the haem oxygenase (HO)/carbon monoxide (CO) pathway may be an important regulator of the inflammatory response, through production of cyclic 3', 5'-monophosphate (cGMP). We have assessed whether HO contributes to the increased production of the pro-inflammatory cytokines TNF-alpha and IL-6 in re-oxygenated rat peritoneal macrophages.Hypoxia/re-oxygenation markedly increased levels of HO-1 mRNA and cGMP. The increase in cGMP was reduced by the HO-1 inhibitor tin-protoporphyrin (SnPP-9) given during re-oxygenation. Hypoxia and re-oxygenation also increased IL-6 and TNF-alpha mRNA expression, as well as IL-6 and TNF-alpha concentrations in the cell supernatant. These increases were nullified by SnPP-9 and by Methylene Blue, an inhibitor of guanylate cyclase, but were not affected by L-NNA, an inhibitor of NO synthesis. The inhibitory effect of SnPP on the synthesis of cytokines was reversed by co-administration of the stable analogue of cGMP, 8-Br-cGMP.Our results indicate that activation of haem oxygenase and of the CO/cGMP pathway is a major stimulus for the synthesis and release of pro-inflammatory cytokines in re-oxygenated macrophages. This pathway may play a central role in pathological situations in which local tissue hypoxia/re-oxygenation triggers a systemic inflammatory response, for example in patients with shock.     

Positive regulatory role of IL-12 in macrophages and modulation by IFN-gamma.

Similar to myeloid dendritic cells, murine macrophages and macrophage cell lines were found to express a surface receptor for IL-12. As a result, peritoneal macrophages could be primed by IL-12 to present an otherwise poorly immunogenic tumor peptide in vivo. Using binding analysis and RNase protection assay, we detected a single class of high affinity IL-12 binding sites (K(d) of approximately 35 pM) whose number per cell was increased by IFN-gamma via up-regulation of receptor subunit expression. Autocrine production of IL-12 was suggested to be a major effect of IL-12 on macrophages when the cytokine was tested alone or after priming with IFN-gamma in vitro. In vivo, combined treatment of macrophages with IFN-gamma and IL-12 resulted in synergistic effects on tumor peptide presentation. Therefore, our findings suggest a general and critical role of IL-12 in potentiating the accessory function of myeloid APC.     

Stimulation of the natural immune system in normal mice by polysaccharide from maitake mushroom

 Maitake D-Fraction is a polysaccharide extracted from the maitake mushroom (Grifola frondosa S.F. Gray). It is a β-glucan with a β-1,6 main chain with β-1,3 branches. Using normal C3H/Hej mice, its effects on the natural immune system, including macrophages, dendritic cells, and natural killer (NK) cells, were investigated. NK cells attack cells infected with pathogens such as bacteria and virus and produce cytokines, such as interferon-gamma (IFN-γ), that can modulate natural and specific immune responses. D-Fraction was administered to the mice intraperitoneally for 3 consecutive days; spleen cells containing macrophages and dendritic cells were then cultured and the culture supernatants were analyzed for IL-12. At the same time, IFN-γ expression in splenic NK cells was investigated. The levels of these cytokines were increased by D-Fraction. To elucidate NK cell activation by D-Fraction, CD69 expression on the surface of activated NK cells was examined, resulting in an increase in CD69-positive ratio for splenic NK cells. These results indicate that D-Fraction stimulates the natural immunity related to the activation of NK cells indirectly through IL-12 produced by macrophages and dendritic cells. Therefore, administration of D-Fraction to healthy individuals may serve to prevent infection. Received: August 1, 2002 / Accepted: February 10, 2003     

Alloantigen-activated lymphocytes from mice bearing a spontaneous "nonimmunogenic" adenocarcinoma inhibit its growth in vivo by recruiting host immunoreactivity

Nylon wool columns eluting lymphocytes from the spleen of mice bearing a clinically evident spontaneous, nonimmunogenic adenocarcinoma of recent origin (TS/A) do not display cytotoxic response, release of lymphokines, and proliferation in vitro against TS/A cells, nor do they inhibit TS/A tumor growth in a Winn-type neutralization assay in vivo. After 5-day co-culture with allogeneic spleen cells from mice differing at multiple minor histocompatibility antigens only, these lymphocytes are still noncytolytic against TS/A cells, whereas they release interferon-gamma, mediate delayed-type hypersensitivity (DTH) reactions, and inhibit TS/A tumor growth in the Winn assay. In the Winn test, alloactivated lymphocytes from TS/A tumor-bearing mice are more effective than those from normal mice on a per cell basis. The induction of this TS/A tumor inhibition ability depends on the presence in the cultures of Thy-1+ lymphocytes. The presence of Lyt-2+ lymphocytes is also important, whereas that of asialo GM1+ is not. The TS/A inhibition in vivo by alloactivated lymphocytes mostly depends on Thy-1+, Lyt-2- and asialo GM- lymphocytes, even though a few Thy- cells are also very efficient tumor inhibitors. The alloactivated lymphocytes inhibit TS/A tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor rejection. TS/A tumor rejection leaves a specific DTH and an immunologic memory resulting in rejection of a second lethal TS/A challenge in a significant number of mice.     

The indispensable role of CCR5 for in vivo suppressor function of tumor-derived CD103+ effector/memory regulatory T cells

CD103 is a marker for identification of effector/memory regulatory T cells (Tregs). CD103(+) Tregs are potent suppressors of tissue inflammation in several infectious diseases, autoimmune diseases, and cancers. However, the underlying mechanisms for this potent suppression ability remain unclear. The current study was designed to clarify this issue. Unexpectedly, we found both CD103(+) and CD103(-) Tregs had similar suppression capacity in vitro. We then chose a murine tumor model for investigation of the in vivo behavior of these Tregs. The suppression ability in vivo against the anti-tumor ability of CD8(+) T cells was restricted to CD103(+) Tregs although both Tregs had equal in vitro suppression ability. In addition, CD103(+) Tregs expressed significantly higher levels of CCR5 than those of CD103(-) Tregs and accumulated more in tumors than did CD103(-) Tregs. Furthermore, blockade of CCR5 signaling, either by CCR5(-/-)CD103(+) Tregs or by CCL5 knockdown tumor, could reduce the migration of CD103(+) Tregs into tumors and impair their in vivo suppression ability. In conclusion, these results indicate that the potent in vivo suppression ability of CD103(+) Tregs is due to the tissue-migration ability through CCR5 expression.     

The regulatory role of adenosine-activated T-lymphocyte subset on the immune response in humans: I. Mitogenic response and production of mediators

Human T lymphocytes, rerosetted with sheep erythrocytes in the presence of adenosine, yield two subpopulations: a major one (E_R), still capable of forming E rosettes; and a minor nonrosetting (E_S) one. The two subpopulations differed in their proliferative responses to various mitogens. E_R cells responded well to galactose oxidase (GO), soybean agglutinin (SBA), and phytohemagglutinin (PHA) but responded poorly to concanavalin A (Con A). The response of E_S cells was poor to GO and SBA, intermediate to PHA, and significantly high to Con A. The different response of E_R and E_S subsets to Con A was not greatly affected by adherent cells, but an enhancing effect on the proliferation of E_S cells to Con A was observed when prostaglandin synthesis was inhibited by indomethacin. Addition of E_S cells to E_R cells in a ratio of 1:5 resulted in an enhanced synergistic effect of Con A-induced proliferation. A soluble mitogenic factor released from Con A-activated T cells appeared involved in this enhanced proliferation. This factor (E_SF) was produced only by the minor T-cell subpopulation which is sensitive to adenosine (E_S). The induction of E_SF was not dependent on the addition of adherent cells and required 72 hr of incubation for its production. E_SF was mitogenic to nonactivated and Con A-activated PBL as well as to T, E_R, and E_S subpopulations. Following incubation of E_R cells with E_SF, a suppressor factor (E_RSF) was produced which abolished the mitogenic activity of E_SF. Differences between these factors and a known mediator like Interleukin-2 (IL-2) and suppressor factors are discussed.     

Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers

Soluble MHC/peptide tetramers that can directly bind the TCR allow the direct visualization and quantitation of Ag-specific T cells in vitro and in vivo. We used HY-D(b) tetramers to assess the numbers of HY-reactive CD8+ T cells in HYTCR-transgenic mice and in naive, wild-type C57BL/6 (B6) mice. As expected, tetramer staining showed the majority of T cells were male-specific CD8+ T cells in female HY-TCR mice. Staining of B6 mice showed a small population of male-specific CD8+ T cells in female mice. The effect of administration of soluble MHC class I tetramers on CD8+ T cell activation in vivo was unknown. Injection of HY-D(b) tetramer in vivo effectively primed female mice for a more rapid proliferative response to both HY peptide and male splenocytes. Furthermore, wild-type B6 female mice injected with a single dose of HY-D(b) tetramer rejected B6 male skin grafts more rapidly than female littermates treated with irrelevant tetramer. In contrast, multiple doses of HY-D(b) tetramer did not further decrease graft survival. Rather, female B6 mice injected with multiple doses of HY-D(b) tetramer rejected male skin grafts more slowly than mice primed with a single injection of tetramer or irradiated male spleen cells, suggesting clonal exhaustion or anergy. Our data highlight the ability of soluble MHC tetramers to identify scarce alloreactive T cell populations and the use of such tetramers to directly modulate an Ag-specific T cell response in vivo.