Interleukin-10 contributes development of macrophage suppressor activities by macrophage colony-stimulating factor, but not by granulocyte-macrophage colony-stimulating factor

Macrophages are known to possess suppressor activities in immune responses. To determine the effects of GM-CSF and M-CSF on the expression of macrophage suppressor activities, monocyte-derived macrophages cultured with GM-CSF (GM-Mphis) were compared with those cultured with M-CSF (M-Mphis) for antigen-specific proliferation and interferon-gamma (IFN-gamma) production by lymphocytes. Both GM-Mphis and M-Mphis equally suppressed lymphocyte proliferation, but only M-Mphis suppressed IFN-gamma production in response to purified protein derivative (PPD). M-Mphis, but not GM-Mphis, released IL-10 not only in the course of macrophage differentiation but also in response to PPD after maturation to macrophages. From the results that (i) exogenous IL-10 suppressed IFN-gamma production, but not proliferation of lymphocytes, and that (ii) neutralizing antibody to IL-10 reversed suppressor activities of M-Mphis on IFN-gamma production, but not lymphocyte proliferation, it appeared that IL-10 was the major factor responsible for suppression of IFN-gamma production. Thus, these results suggest that only M-CSF augments IL-10-dependent suppressor activity of macrophages on IFN-gamma production and that both GM-CSF and M-CSF induce IL-10-independent macrophage suppressor activity on lymphocyte proliferation.     

c-Myc sensitizes cells to tumor necrosis factor-mediated apoptosis by inhibiting nuclear factor kappa B transactivation

Nuclear factor kappaB (NF-kappaB) plays a key role in suppression of tumor necrosis factor (TNF)-mediated apoptosis by inducing a variety of anti-apoptotic genes. Expression of c-Myc has been shown to sensitize cells to TNF-mediated apoptosis by inhibiting NF-kappaB activation. However, the precise step in the NF-kappaB signaling pathway and apoptosis modified by c-Myc has not been identified. Using the inducible c-MycER system and c-Myc null fibroblasts, we found that expression of c-Myc inhibited NF-kappaB activation by interfering with RelA/p65 transactivation but not nuclear translocation of NF-kappaB. Activation of c-Myc promoted TNF-induced release of cytochrome c from mitochondria to the cytosol because of the inhibition of NF-kappaB. Furthermore, we found that NF-kappaB-inducible gene A1 was attenuated by expression of c-Myc and that the restoration of A1 expression suppressed c-Myc-induced TNF sensitization. Our results elucidate the molecular mechanisms by which c-Myc increases cell susceptibility to TNF-mediated apoptosis, indicating that c-Myc may exhibit its pro-apoptotic activities by repression of cell survival genes.     

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Production of granulocyte colony-stimulating factor by head and neck carcinomas

Detectable levels of G-CSF by enzyme-linked immunosorbent assay (ELISA) were found in sera of 4 out of 15 patients with head and neck carcinomas. Also cells prepared from the tumors of these 4 patients secreted G-CSF. The supernatants of cells derived from all 15 patients did not contain granulocyte-monocyte CSF, monocyte CSF, tumor necrosis factor-, transforming growth factor- ₁, epidermal growth factor, interleukin (IL)-1 and IL-6. These findings suggest that leukocytosis in patients with carcinomas might be due to the production of G-CSF by tumor cells.Abbreviations CSF colony stimulating factor - EGF epidermal growth factor - ELISA Enzyme-linked immunosorbent assay - G granulocyte - GM granulocyte-monocyte - IL interluekin - M monocyte - TGF transforming growth factor - TNF tumor necrosis factor     

The role of NK cells in tumor growth assessed by induction of their suppressor cells in Japanese quails

In Japanese quails treated with chicken amniotic fluid (ChAmF) which had been previously shown to induce suppressor cells to natural killer (NK) cells, tumors appeared with shortened incubation periods after inoculation with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) compared with untreated quails. The tumors in ChAmF-treated quails subsequently grew in a similar pattern to those in untreated quails, whereas by challenging with a lower dose of the virus, enhanced tumor growth was observed as well as earlier onset of tumors in ChAmF-treated quails than in untreated ones. This enhancing effect on tumor growth due to suppression of NK-cell activity was transferred to normal quails with spleen cells obtained from ChAmF-treated quails, since RSV-induced tumors appeared earlier in the recipients of ChAmF-treated spleen cells than in those of untreated spleen cells. These findings show that suppression of NK-cell activity by ChAmF administration rendered quails higher susceptibility to tumor induction by SR-RSV challenge. In other words, NK-cell activity was strongly suggested to contribute to the early protection against tumor growth in the system of Rous sarcoma in Japanese quails.     

Prostaglandin E2-dependent induction of granulocyte-macrophage colony-stimulating factor secretion by cloned murine helper T cells

PG are known to inhibit T cell proliferation, at least in part by suppressing IL-2 production, but effects of PG on the production of other lymphokines have not been well studied. We have found that PGE2 and PGE1, but not PGF2 alpha, inhibit both proliferation and production of granulocyte-macrophage (GM)-CSF by murine TH clones stimulated with Ag or anti-CD3 antibody. Thus, signals generated via the Ag receptor:CD3 complex were inhibited by PGE. Most interesting, however, was the finding that PGE2 and PGE1 could act synergistically with IL-2 for the induction of GM-CSF in some TH1 clones. Dependence on PGE2 for this response was not found in all clones, as some TH1 cells could produce GM-CSF after IL-2 alone, and some cells did not produce GM-CSF even in the presence of PGE2 and IL-2. These observations indicate that there is a subset of TH1 cells receptive to a stimulating activity of PGE2 in the presence of IL-2. PGE2 is known to elevate cAMP levels in T cells. Therefore, we tested whether other agents known to increase cAMP, such as forskolin and cholera toxin, could act in conjunction with IL-2 to induce GM-CSF secretion. As was found with PGE2, these compounds also induced GM-CSF activity in the presence of IL-2, suggesting a critical role for cAMP in this process. Overall these data indicate that the requirements for activation of GM-CSF secretion vary among individual T cells. Most importantly they provide the first evidence that E-series PG are positive signals for lymphokine induction in certain T cells, whereas simultaneously acting as negative signals limiting proliferation. This result also suggests that treatment with anti-inflammatory drugs that decrease PGE2 concentrations may inhibit lymphokine secretion normally stimulated by this pathway.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Production of granulocyte/macrophage colony-stimulating factor by cultured astrocytes

We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide (LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3- and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GM-CSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF.     

Intraclonal diversity of fibrosarcoma cells for the production of macrophage colony-stimulating factor and granulocyte colony-stimulating factor

A new cell line was established from fibrosarcoma that had spontaneously developed in a mouse. The cells were maintained growing in culture for two years and constantly produced both macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). Cloning of the cells by anchorage-independent colony formation gave subclones showing the activity of producing M-CSF and G-CSF in different proportions, whereas no subclone produced G-CSF without producing M-CSF simultaneously. Recloning of the bipotential subclones again gave clonal derivatives producing two types of CSF in various proportions. The observed heterogeneity of the cloned cells seems to be an epigenetic phenomenon, because the cells resumed the G-CSF producing activity in the absence of cell proliferation. After equilibrium was achieved, all of the subclones produced both M-CSF and G-CSF nearly in equal proportions. Tumorigenic and leukocytosis-inducing activity of the cloned cells was nearly comparable with the activity of the original tumor cells.     

Synergism between stem cell factor and granulocyte-macrophage colony-stimulating factor on cell proliferation by induction of cyclins

Synergism between stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be essential for hematopoietic cell proliferation. Since HML-2 cells proliferate exponentially in the presence of SCF and GM-CSF together, we analyzed the molecular mechanism of the interaction between these two factors in the cells. An immediate-early gene product, c-myc, was additively upregulated in HML-2 cells by addition of a combination of SCF and GM-CSF. c-myc antisense oligonucleotides effectively suppressed cell proliferation and downregulated the induction of D3, E, A, and B cyclins in HML-2 cells stimulated with the two-factor combination. HML-2 cells arrested at the G0/G1 phase with SCF alone and expressed modest amounts of c-myc and cyclin D3, but not cyclin E. With GM-CSF treatment alone, the cells could not progress to the G2/M phase and expressed c-myc, cyclin D3 and cyclin E but not cyclins A or B. The addition of the counterpart cytokine resulted in cell cycle completion by induction of the deficient cyclins. Taken together, it appears that the induction of c-myc is an indispensable event in the proliferation of HML-2 cells and that the cytokines SCF and GM-CSF interact reciprocally for expression of all cyclins required for cell cycle progression.     

Concomitant induction of cytotoxic and suppressor cells: modulation by theophylline

Previous studies in man have shown that T cells with suppressor activity were mainly found among a subset bearing Fc receptors for IgG (T_γ). Recently, we found that virus-induced cytotoxic effector cells were also found predominantly among T_γ cells. In the present studies, we present evidence that similar, possibly overlapping T-cell populations can mediate both suppressor and cytotoxic activities when sensitized in vitro with virus-infected cells. In fact, both activities are found within the positively selected T_γ subset, but not in the T_γ-depleted population; both activities are abolished by irradiation but not by treatment with mitomycin C; a 1-hr exposition to theophylline at the onset of sensitization enhances both cytotoxic and suppressor activities. The data suggest that development of antiviral cell-mediated immune responses in vivo may also be accompanied by a concurrent induction of nonspecific suppressor cells. Such suppressor activity may play a role in the depressed cellular immune responsiveness which is associated with several systemic virus infections.     

Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells

The role of Ca2+ in cell-mediated cytotoxicity has been the subject of many investigations and both Ca2+-dependent and -independent pathways have been reported. TNF was suggested to play a role in NK and macrophage cell-mediated cytotoxicity. We assumed that its role in target cell lysis might take place by a Ca2+-independent mechanism. This hypothesis was investigated in assays of rTNF-mediated lysis of tumor target cells. Extracellular Ca2+ depletion by the calcium chelator EGTA (2 mM and 5 mM) and blocking of intracellular Ca2+ mobilization by 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride did not inhibit TNF-mediated tumor cell lysis. Furthermore, blocking of Ca2+ influx in the presence of the Ca2+ channel blocker Verapamil did not inhibit TNF-mediated tumor cell lysis. Previous reports showed that lysis of sensitive tumor cells by TNF is preceded by binding of TNF to TNF receptors, internalization, and DNA degradation. These events were tested in the absence of Ca2+. Treatment with Ca2+ inhibitors did not affect binding of 125I-TNF to target cells. Also TNF induced the fragmentation of cellular DNA in target cells without extracellular or intracellular Ca2+. These findings demonstrate that the mechanism of TNF-mediated tumor cell lysis does not depend on intracellular or extracellular Ca2+ and that events associated with target cell lysis can also function in the absence of Ca2+. Thus, our findings support the contention of a Ca2+-independent lytic pathway in which secreted or membrane-bound TNF may interact with the target cells and ultimately result in DNA degradation and target cell lysis.     

Production of soluble suppressor factor by spleen cells from mice immunized with type III pneumococcal polysaccharide

Supernatant fluid (SF) derived from spleen cell cultures, obtained from mice 16 hr after immunization with 0.5 microgram of Type III pneumococcal polysaccharide (SSS-III), suppressed the antibody response when SF was given (i.v.) 3 hr before immunization with SSS-III. Such suppression was antigen specific and could be reproduced by SF derived from cultures of T cells from mice immunized with SSS-III (0.5 microgram) or by SF derived from cultures of spleen cells from mice primed with a subimmunogenic dose of SSS-III (0.005 microgram). Adsorption of SF with SSS-III covalently bound to a Sepharose 4B column did not alter the ability of SF to suppress the SSS-III-specific antibody response. However, adsorption of SF with Ig+ (B) cells from mice immunized with 0.5 microgram SSS-III completely removed the suppressive activity. Significant (p less than 0.05) suppression of the antibody response was observed only when SF was administered (i.v.) 24 hr before to 24 hr after immunization with 0.5 microgram of SSS-III. These results suggest that suppressor T cells generated in response to SSS-III function by releasing a soluble factor(s) that binds to determinants on B cells rather than antigen; this soluble factor(s) acts directly on antigen-stimulated B cells or inhibits the induction of amplifier T cells.