Regulation of macrophage activation markers by IL-4 and IFN-gamma is subpopulation-specific

We have addressed the differential regulatory properties that IFN-gamma and IL-4 exert on macrophage (M phi) subpopulations. For this purpose, Thyoglicolate-, Peptone-, and Con A-elicited M phi, as well as bone marrow-derived M phi and P388D1 cells, were cultured in the presence of either IFN-gamma or IL-4. The expression of LFA-1, Mac-1, and Mac-2 after this treatment was studied by FACS analysis. We have found that these surface molecules are differentially modulated by the two lymphokines, depending on the M phi subpopulation studied. Mac-1 is upregulated only in Thyoglicolate-elicited cells after treatment with IFN-gamma, while no change in the expression of Mac-2 was observed in any of the groups. LFA-1 is upregulated by IFN-gamma in Thyoglicolate- and bone marrow-derived M phi and P388D1 cells, while IL-4 does not induce LFA-1 on these cells. Interestingly, however, we have observed the reverse situation on Con A-elicited M phi, where a strong induction of LFA-1 is achieved by treatment of the cells with IL-4, while IFN-gamma does not modify the expression of this antigen. Our results obtained with the lymphokine-stimulated M phi are interpreted in the context of functionally induced M phi subpopulations, which might be regulated by either Th1 or Th2 CD4+ T cells. Thyoglicolate-elicited M phi may represent the in vitro equivalent of a M phi subpopulation regulated in vivo by Th1 cells while Con A-elicited M phi could be the equivalent of a subpopulation regulated by Th2 cells.     

Leukotriene C4 produced by a human T-T hybridoma suppresses Ig production by human lymphocytes

Multiple signals are involved in the regulation of Ig production by human B lymphocytes. Leukotrienes, especially LTB4, have been shown to inhibit Ig production by increasing the number and function of suppressor lymphocytes. Production of leukotrienes has been demonstrated by mast cells, basophils, eosinophils, polymorphonuclear leukocytes, monocytes, and macrophages. In this paper we demonstrate that a human T-T hybridoma grown at 5 x 10(5) cells/ml constitutively produces 5 ng/ml of LTC4. Furthermore, we demonstrate that either the supernatant from this hybridoma containing 0.5 to 10 ng/ml LTC4 or purified LTC4 in the range of 0.5 to 5 ng/ml can suppress 50 to 70% of Ig production by unfractionated human mononuclear cells, by normal human cells stimulated with Staphylococcus aureus Cowan I and B cell differentiation factors, and by the EBV-transformed B cell line SKW.6 in the presence of B cell differentiation factors. Thus, LTC4 can have direct effects on B cells and may have a role in normal B cell regulation.     

Characterization of monoclonal B cell growth factor (BCGF) produced by a human T-T hybridoma

We previously demonstrated the development of a cloned human T cell hybridoma that secretes B cell growth factor (BCGF) in the absence of demonstrable interleukin 2 or B cell differentiation factor. Sephadex gel filtration chromatography demonstrated the m.w. of this factor to be 18 to 20K. The present studies were performed to further characterize the biochemical properties of the molecule and to determine its target cell specificity. Temperature stability studies showed the monoclonal BCGF to be stable at 37 degrees C for 12 hr and at 70 degrees C for 15 min; however, most (93%) of the activity was lost after incubation at 70 degrees C for 30 min. Aliquots of hybridoma supernatant were exposed to buffer solutions with variable pH with no diminution in activity over a pH range of 4.0 to 10.0 BCGF activity was not affected by 2-mercaptoethanol, neuraminidase, or nucleic acid denaturing enzymes. In contrast, all activity was destroyed by 10 M urea, trypsin, and chymotrypsin. Chromatofocusing demonstrated the isoelectric point of BCGF to be 6.3 to 6.6. Finally, absorption experiments demonstrated that BCGF activity was absorbed by large, activated B cells. Mitogen-stimulated T cell blasts, small resting B cells, and CESS cells failed to absorb BCGF activity from the hybridoma supernatant. These and future studies with purified monoclonal human BCGF should enhance our understanding of its immunochemical properties and of its role in the immunoregulation of human B cell responses.     

Activation of murine macrophages by hydrogen peroxide

Hydrogen peroxide at concentrations from 0.1 to 20 μM enhances phagocytosis and oxidative burst of murine peritoneal macrophages. The activation of these macrophage functions is paralled by prolonged hyperpolarization and a transient increase in cytoplasmic free calcium concentration. All the effects are dose- and time-dependent. The results obtained for H₂O₂ are compared with those for a natural activator, peptide N-formyl-methionyl-leucly-phenylalanine. The data demonstrate the ability of small doses of hydrogen peroxide to stimulate macrophages through the intracellular mechanisms of ion transduction.     

Down-regulation of murine collagen-induced arthritis by a T cell hybridoma

T cell hybridoma cell lines were generated by somatic cell fusion of BW 5147 myeloma cells and splenic cells from mice suppressed for collagen-induced arthritis (CIA). Two cell lines were characterized for their cell surface phenotype, antigen recognition and capacity to down-regulate the erythema and edema associated with CIA. Cell line T101N was determined to portray the cell surface phenotype Ly1+2- L3T4- Thy1+ by a direct binding assay. Cell line T104B1 was determined to express only the Thy1+ alloantigen. Panning studies, measurement of IL-2 production in vitro and the suppression of antibodies to type I and type II collagen in vivo indicate that the hybridoma cells are not isotype specific in their recognition of the polymorphic interstitial collagens. Down-regulation of the erythema and edema of CIA occurred on injection of 1 X 10(5) T101N cells but not T104B1 cells. Histology of the tarsus region of the hind paw of CIA mice 33 days after the administration of T101N cells showed contrasting histopathology compared to that of CIA mice. The joints of CIA mice given T101N cells showed aligned articular surfaces resembling normal joint structure and only residual pannus. The data indicate that collagen-specific cloned T cell lines can modulate the gross pathology and joint architecture of joints exhibiting CIA.     

Monoclonal antibodies block murine IL-4 receptor function.

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.     

Murine recombinant IL-4 is a bifunctional regulator of macrophage growth induced by colony-stimulating factors.

Murine peritoneal exudate macrophage (PEM) coexpress receptors for both granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) and can be induced by both factors, either alone or in combination, to undergo extensive proliferation in vitro. In this study the effect of murine rIL-4 (MurIL-4) on the proliferation of PEM was examined. MurIL-4 alone did not support macrophage proliferation but prolonged their survival in vitro. When MurIL-4 was combined with human (Hu)rM-CSF, it enhanced the proliferative response of PEM to rHuM-CSF in a dose-dependent manner, reaching a maximum at approximately 10 ng/ml. Contrarily, MurIL-4 suppressed the proliferative response of PEM to MurGM-CSF. Receptor binding assays using radiolabeled ligands showed that MurIL-4 selectively enhanced the expression of M-CSF receptors; suggesting that at least part of the synergistic effect of MurIL-4 is mediated at the receptor level. Of relevance to this effect is the finding that MurIL-4 greatly promoted the responsiveness of PEM to low concentrations of HurM-CSF. Unlike M-CSF receptors, however, MurIL-4 treatment failed to modulate the levels of GM-CSF receptors in PEM. The proliferative responses of PEM to both MurGM-CSF and HurM-CSF could be inhibited by MurIFN-gamma with similar sensitivity. This inhibitory effect of MurIFN-gamma was partially neutralized by MurIL-4 in cultures containing HurM-CSF but not those containing MurGM-CSF. This study demonstrates that IL-4 is involved directly in the regulation of macrophage production by modulating their responsiveness to various cytokines.     

Regulation of murine B cell Thy-1 expression by IL-4, IFN-gamma, and CD4+ T cell subsets

Interleukin 4 (IL-4) induces the expression of membrane Thy-1 on the vast majority of lipopolysaccharide (LPS)-stimulated normal murine B cells in vitro. This induction is inhibited by interferon-gamma (IFN-gamma). IL-4 and IFN-gamma are required late in culture to effect maximal induction and inhibition of Thy-1 expression by LPS- or LPS + IL-4-stimulated B cells, respectively. IFN-gamma suppresses IL-4-induced Thy-1 expression by inhibiting the induction of steady-state levels of Thy-1-specific mRNA. Three distinct CD4+ Th2 clones, through their release of IL-4, induce B cells to express high levels of Thy-1, by 24 hr, in striking contrast to the 3 days required to induce Thy-1 expression after stimulation with LPS and IL-4. This induction is abrogated by the addition of IFN-gamma. B cells stimulated with three distinct Th1 clones (IFN-gamma- and IL-2-producing) exhibit a modest, non-IL-4-dependent, expression of Thy-1. In contrast to intrinsic expression of Thy-1 by Th2-stimulated B cells. Thy-1 expressed by Th1-stimulated B cells is acquired, having the allotype specificity of the stimulating T cell.     

IL-10 inhibits mitogen-induced T cell proliferation by selectively inhibiting macrophage costimulatory function.

IL-10, a newly designated cytokine primarily produced by the Th2 subset of CD4+ T lymphocytes and Ly-1+ B lymphocytes, has recently been hypothesized to inhibit cytokine production by Th1 T cell clones by blocking accessory cell- (AC) dependent costimulatory function. To evaluate the effect of IL-10 on Con A-induced proliferative responses of resting murine T cells, purified T cells were cultured with different types of AC. The addition of IL-10 produced a 70 to 90% inhibition of resting T lymphocyte proliferation when purified populations of macrophages were used as AC, but had no effect on the AC function of T-depleted spleen cells, activated B cells, dendritic cells, or L cells. The inhibitory effects of IL-10 were inversely related to the concentration of mitogen and could be reversed by the addition of the neutralizing anti-IL-10 mAb, SXC1. The inhibition of macrophage AC function was not related to the induction of a suppressor cytokine as stimulation by mixtures of macrophages and limiting numbers of dendritic cells was not inhibited. The decrease in proliferative responses was primarily secondary to inhibition of IL-2 production although the failure of exogenous IL-2 to completely reconstitute the response suggested that IL-10 may also exert inhibitory effects on the induction of expression of a functional IL-2R. These results are most consistent with a model in which IL-10 inhibits the induction of expression on macrophages of a critical costimulatory molecule that may be constitutively expressed on other types of AC.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

Regulation of macrophage populations. II. Synthesis and expression of Ia antigens by peritoneal exudate macrophages is a transient event

We have evaluated the biosynthesis and surface expression of I-A antigens by peritoneal macrophages and found that both events terminated during the 1st day in culture, in contrast to the undiminished synthesis and expression of H-2K antigens. This pattern was observed regardless of the means by which the macrophages were elicited, but was subject to modulation for a limited period of time in vitro: phagocytic stimuli were able to augment both I-A synthesis and expression. The loss of I-A and the re-expression after phagocytosis were both reflected in the stimulatory capacity of these macrophages in the mixed leukocyte reaction. Moreover, we found that I-A-bearing macrophages were lost from the exudate in vivo after irradiation. Our data suggest that, as in vitro, this phenomenon is due to the transition of individual macrophages from I-A-positive to I-A-negative, and that constant renewal is required to maintain the I-A-bearing subset in vivo.     

IL-1 beta is secreted by activated murine macrophages as biologically inactive precursor

IL-1 alpha and IL-beta are distinct cytokines, produced by activated macrophages. The temporal sequence in the processing and secretion as well as the mechanism(s) by which IL-1 is secreted from the cells remain undefined. Here we have studied the production of IL-1 from murine macrophages after stimulation with LPS or Listeria monocytogenes by two distinct methods: i) immunoprecipitation of radio-labeled IL-1 peptides from culture supernatants, and ii) determination of IL-1 activity by neutralization with monospecific antisera to either form of IL-1. We confirmed that precursor and mature forms of both IL-1 alpha and IL-1 beta can be detected in the culture supernatants after stimulation of the macrophages with 10 to 20 micrograms LPS/ml but, in addition, we report the novel finding that IL-1 beta is exclusively secreted in its unprocessed precursor form after stimulation of the cells with either 0.5 to 1 microgram LPS/ml or with L. monocytogenes. Exposure of the cells to increasing amounts of LPS led to the appearance of a 20-kDa IL-1 beta peptide in the culture supernatants concomitant with the release of a processing activity for the IL-1 beta precursor. These data therefore suggest that, in a first step, IL-1 beta is secreted as an unprocessed precursor protein that in a second, postsecretory step is cleaved by a LPS-inducible protease, thus generating the 20-kDa IL-1 beta peptide. The latter represents the biologically active IL-1 beta inasmuch as the generation of IL-1 beta activity in the culture supernatants strictly correlated with the appearance of the 20-kDa IL-1 beta peptide.     

Inhibition of lymphoid cell growth by a lipid-like component of macrophage hybridoma cells

Previous studies have shown that plasma membranes of murine lymphocytes and lymphoid tumor cells can reversibly inhibit the growth of both normal and transformed lymphocytes. The inhibitor can be extracted with organic solvents and has properties consistent with it being a lipid or lipid-like component of the membrane. This report identifies a series of cloned macrophage hybridoma cell lines, obtained by fusion of splenic adherent cells and the P388D1 line, which have very high levels of lipid-like growth-inhibitory molecules. Furthermore, a survey of seven cloned lines indicated that the macrophages fell into two distinct groups with regard to their level of growth-inhibitory activity. Group 1 lines had little or no inhibitory activity when cells were examined for their effect on a B lymphocyte proliferative response. Organic extracts from these macrophages had inhibitory activity (on a per cell basis) comparable to that seen with extracts of the P388D1 parental cell line and lymphoid tumor cells. In contrast, relatively low numbers of Group 2 macrophages could profoundly inhibit B macrophage proliferation. The growth-inhibitory activity was quantitatively recovered in organic extracts of the macrophages. Although the precise nature of the lipid moiety remains undefined, the data argue against the involvement of oxidized cholesterol. These findings indicate that lipid-like inhibitors of cell growth are present and functional in these macrophage cell lines. In addition, the results demonstrate that the inhibitory activity found in plasma membranes and liposomes is present and active in the membranes of intact cells, which is in contrast to the possibility that the inhibitor is an artifact generated during subcellular fractionation. Thus, the inhibitor is likely to have a physiologic role in growth control and in macrophage-mediated immunoregulation, probably acting via a mechanism involving cell-cell contact.     

Characterization of IL-2 receptor expression and function on murine macrophages

This study was designed to examine the expression and function of IL-2R on murine macrophages. We used a model system of murine macrophage cell lines (ANA-1 and GG2EE) that was established by infecting normal murine bone marrow-derived cells with the J2 (v-raf/v-myc) recombinant murine retrovirus. ANA-1 macrophages did not constitutively express detectable levels of mRNA for the p55, IL-2R alpha. However, a brief exposure to IFN-gamma was sufficient to induce IL-2R alpha mRNA in ANA-1 macrophages. Flow cytometric analysis indicated that ANA-1 macrophages expressed low constitutive levels of IL-2R alpha on their cell surface that were augmented after treatment of the cells with IFN-gamma. Affinity binding and cross-linking of [125I]IL-2 to ANA-1 macrophages demonstrated that IL-2R alpha and the p70-75, IL-2R beta were both present on ANA-1 macrophages constitutively. IFN-gamma increased the expression of IL-2R alpha on ANA-1 macrophages but did not increase the expression of IL-2R beta on these macrophages. Although IL-2 alone did not induce the tumoricidal activity of ANA-1 macrophages, IL-2 acted synergistically with IFN-gamma to induce macrophage tumoricidal activity. These data demonstrate the expression of IL-2R on murine macrophage cell lines and establish the role of IL-2 as a costimulator of macrophage-mediated tumoricidal activity.     

Bone marrow stromal cell regulation of B-lymphopoiesis. I. The role of macrophages, IL-1, and IL-4 in pre-B cell maturation

Our experiments have addressed regulation of B lymphocyte formation by bone marrow stromal cells. Stromal cells appear to produce a regulatory factor that acts at the pre-B cell stage to induce the expression of Ig L chains and surface Ig. Bone marrow stromal cell conditioned medium was found to contain this factor and the active component was partially purified by HPLC. This stromal cell-derived factor had a m.w. between 16,000 and 20,000, was specifically neutralized by anti-IL-4 mAb, 11B11, and enhanced the proliferation of anti-mu-stimulated B cells. We also found that rIL-4 induced B cell formation in culture. In our studies, IL-1 had no direct effect on pre-B cell maturation, however, IL-1 was found to stimulate the production of IL-4 by both heterogeneous bone marrow stromal cells and a cloned stromal cell line, SCL-160. These effects of IL-1 on factor production by stromal cells were duplicated by the addition of bone marrow-derived macrophages to SCL-160 cells. We conclude that stromal cell-derived IL-4 is a physiologic stimulator for B cell generation. In addition, macrophages appear to play a role in B cell formation by regulating the production of IL-4 by stromal cells via the secretion of IL-1.     

Over expression of IL-10 by macrophages overcomes resistance to murine filariasis

Individuals infected with parasitic helminths are able to tolerate the presence of parasites for considerable time without clinical pathology. Immunosuppressive responses induced by the filarial parasite are considered responsible for this long-lasting relationship, inuring to the benefit of both parasite and host. In order to directly link IL-10 with parasite survival, we infected mice, in which over expression of IL-10 was restricted to macrophages under control of the CD68 promoter (macIL-10tg), with Litomosoides sigmodontis. IL-10 overexpression by macrophages led to increased susceptibility with a significantly higher number of adult worms. Most profound, IL-10 overexpression was sufficient to convert resistant FVB wild-type mice towards a patent phenotype, since microfilariae were exclusively found in macIL-10tg mice. These findings were associated with reduced Th2 cytokine production in macIL-10tg mice. Expression of arginase-1, Ym1 and Fizz1, genes that are found strongly expressed in murine alternatively activated macrophages, were detected in macIL-10tg mice. Thus, IL-10 produced by macrophages with characteristics of alternative activation can overcome resistance and allow full patency in murine filariasis.     

Induction of class I and class II MHC antigen expression on murine bone marrow-derived macrophages by IL-4 (B cell stimulatory factor 1)

We have studied the effects of IL-4 (B cell stimulatory factor 1) on the expression of MHC gene products in normal bone marrow-derived macrophages, peritoneal macrophages, and the myelomonocytic cell line WEHI-3. Using both IL-4-containing T cell supernatant and rIL-4, we have observed significant induction of both class I and class II MHC surface expression (about 1.5- to 4-fold increase) in 2-, 3-, and 4-day cultures of bone marrow-derived macrophages. This induction was also apparent at the mRNA level as assessed by Northern blot analysis using A beta, E alpha, and class I probes. Kinetic analysis revealed that induction of class II mRNA by IL-4 was slower than induction by IFN-gamma, requiring 48 h before a significant increase was noted. The magnitude of MHC induction by IL-4 was not as great as that seen with IFN-gamma, which was found to increase surface expression of MHC antigens two- to eightfold. IL-4 also differs from IFN-gamma in the repertoire of macrophages responsive to it. IL-4 was unable to induce class I or class II expression in either thioglycolate-elicited peritoneal macrophages or WEHI-3 cells whereas IFN-gamma induced MHC antigen expression on both cell types under the same conditions. These data demonstrate that IL-4 is capable of inducing both class I and class II MHC gene products in some, but not all, macrophages.