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Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-gamma induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-gamma-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-gamma-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1(-/-) macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-gamma-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-gamma signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-gamma-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4(+) T cells.

Induction of a Th1 immune response against viral infection of the CNS is important in contributing to viral clearance. The present studies demonstrate a role for the T cell chemoattractant chemokine Mig (monokine induced by IFN-gamma) in contributing to a Th1 response against mouse hepatitis virus infection of the CNS. Analysis of the kinetics of Mig expression revealed mRNA transcripts present at days 7 and 12 postinfection (p.i.) but not early (day 2) or late (day 35) in the infection. To determine functional significance, mouse hepatitis virus-infected mice were treated with anti-Mig antisera, and the severity of disease was evaluated. Such treatment resulted in a marked increase in mortality that correlated with a >3 log increase in viral burden within the brains as compared with control mice treated with normal rabbit serum. Anti-Mig-treated mice displayed a significant decrease (p < 0.005) in CD4(+) and CD8(+) T cell recruitment into the CNS as compared with normal rabbit serum-treated mice. In addition, anti-Mig treatment resulted in a significant decrease (p < 0.05) in levels of IFN-gamma and IFN-beta that coincided with increased (p < 0.02) expression of the anti-inflammatory Th2 cytokine IL-10 within the CNS. Collectively, these data indicate that Mig is important in contributing to host defense by promoting a protective Th1 response against viral infection of the CNS.

The contribution of the T cell chemoattractant chemokine IFN-inducible protein 10 (IP-10) in host defense following viral infection of the CNS was examined. IP-10 is expressed by astrocytes during acute encephalomyelitis in mouse hepatitis virus-infected mice, and the majority of T lymphocytes infiltrating into the CNS expressed the IP-10 receptor CXCR3. Treatment of mice with anti-IP-10 antisera led to increased mortality and delayed viral clearance from the CNS as compared with control mice. Further, administration of anti-IP-10 led to a >70% reduction (p

Macrophage inflammatory protein 1alpha (MIP-1alpha) promotes natural killer (NK) cell inflammation in livers during murine cytomegalovirus (MCMV) infections, and NK cell-produced interferon gamma (IFN-gamma) contributes to defense against MCMV infections. A specific role for local NK cell IFN-gamma production, however, has not been established. The importance of MIP-1alpha and NK cell-produced IFN-gamma in shaping endogenous immune responses and defense in different compartments was examined. MIP-1alpha deficiency profoundly decreased resistance to MCMV and was associated with dramatically reduced NK cell accumulation and IFN-gamma production in liver. MIP-1alpha-independent IFN-gamma responses were observed in serum and spleen, and infection-induced elevations in blood NK cell populations occurred in absence of the factor, but peak liver expression of another chemokine, the monokine induced by IFN-gamma (Mig), depended upon presence of MIP-1alpha, NK cells, and IFN-gamma. The Mig response was also important for viral resistance. Thus, serum cytokine responses are insufficient; MIP-1alpha is critical for NK cell migration and IFN-gamma delivery to mediate protection; and Mig induction in tissues is a downstream protective response resulting from the process. These results define a critical chemokine-to-cytokine-to-chemokine cascade required for defense during a viral infection establishing itself in tissues.

IFNgamma is a functionally pleiotropic cytokine which shows considerable potency in promoting anti-tumor functions in vivo. Despite limited efficacy when delivered systemically either to experimental animals or patients, IFNgamma appears to play an important and perhaps critical role in directing the development of immune-mediated tumor destruction when expressed within the tumor bed. This has been demonstrated both by use of tumor cells transduced to express IFNgamma and by the use of IL-12 which is able, at least is murine models, to promote an IFNgamma-dependent, T cell mediated anti-tumor response. Recent studies indicate that the therapeutic efficacy of IFNgamma in tumor models depends critically upon the ability of the tumor cells themselves to respond to IFNgamma. Though IFNgamma is able to induce anti-viral activity and has direct anti-proliferative effects on some tumor cell lines, immunomodulatory function also appears to be an important component of its anti-tumor action. This is mediated through the action of several different classes of IFNgamma-inducible gene expression which control antigen processing and presentation, leukocyte trafficking, and indirect tumor cytotoxicity.

Interleukin 12 (IL-12) is known to play an important role in the development of an antitumor response. Its activity has been shown to be dependent upon the intermediate production of IFN-gamma and the influx into the tumor of CD8 lymphocytes. In a murine model, tumor regression induced by IL-12 treatment correlated with IFN-gamma, IP-10, and Mig expression in the tumor bed and was abrogated by antibodies to both chemokines. Here we examined the effects of rHuIL-12 on IFN-gamma and CXC chemokine gene expression in patients with renal cell carcinoma (RCC) in an attempt to determine whether a similar series of molecular events leading to IL-12-mediated tumor regression in mice is also detectable in humans. As in the murine RENCA model, cultured RCC cells themselves could be induced by IFN-gamma to synthesize IP-10 and Mig mRNA. Explanted RCC produced IFN-gamma and IP-10 mRNA in response to IL-12 treatment, which was consistent with the finding that biopsied RCC tumors from IL-12-treated patients also variably expressed augmented levels of those molecules after therapy. Although Mig mRNA was present in the majority of biopsied tumors prior to treatment, both the Mig and IP-10 chemokines as well as IFN-gamma were induced in the peripheral blood mononuclear cells of IL-12-treated patients. Skin biopsies of IL-12-treated patients also all synthesized IP-10 mRNA. This study demonstrates that recombinant human IL-12 therapy of patients with RCC has the potential to induce the expression of gene products within the tumor bed that may contribute to the development of a successful antitumor response.

IL-4 suppresses the IFN-gamma-induced expression of the IFN regulatory factor-1 (IRF-1) gene, and this suppression is attenuated by increasing the amount of IFN-gamma. The effects of IFN-gamma and IL-4 on transcription of a reporter gene under control of a 1.3-kb fragment from the IRF-1 gene promoter or the STAT binding element (SBE) from this gene in the context of a heterologous promoter are similar to their effects on the endogenous IRF-1 gene. IFN-gamma-dependent transcription of reporter gene is suppressed by IL-4, but IL-4 alone has no trans-activating function. IL-4 treatment does not inhibit the tyrosine phosphorylation or nuclear translocation of IFN-gamma-activated STAT1. Rather, IFN-gamma and IL-4 independently activate STAT1 and STAT6, respectively, and both proteins bind to the IRF-1 SBE in homodimeric form. The affinity of STAT1 for the IRF-1 SBE is higher than the affinity of STAT6, as measured by competition with unlabeled oligonucleotide. These observations suggest that IL-4 may suppress IFN-gamma-stimulated transcription of the IRF-1 gene by activation of STAT6, which can compete with STAT1 for occupancy of the IRF-1 SBE when STAT1 levels are low. Suppression may be attenuated as the quantity of STAT1 relative to that of STAT6 increases in cells treated with increasing amounts of IFN-gamma and displaces STAT6.

Interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) cooperate to induce the expression of many gene products during inflammation. The present report demonstrates that a portion of this cooperativity is mediated by synergism between two distinct transcription factors: signal transducer and activator of transcription 1 (STAT1) and nuclear factor kappaB (NF-kappaB). IFNgamma and TNFalpha synergistically induce expression of mRNAs encoding interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1, Mig (monokine induced by gamma-interferon), and RANTES (regulated on activation normal T cell expressed and secreted) in normal but not STAT1-deficient mouse fibroblasts, indicating a requirement for STAT1. Transient transfection assays in fibroblasts using site-directed mutants of a 1.3-kilobase pair sequence of the IRF-1 gene promoter revealed that the synergy was dependent upon two sequence elements; a STAT binding element and a kappaB motif. Artificial constructs containing a single copy of both a STAT binding element and a kappaB motif linked to the herpes virus thymidine kinase promoter were able to mediate synergistic response to IFNgamma and TNFalpha; such response varied with both the relative spacing and the specific sequence of the regions between these two sites. Cooperatively responsive sequence constructs bound both STAT1alpha and NF-kappaB in nuclear extracts prepared from IFNgamma- and/or TNFalpha-stimulated fibroblasts, although binding of individual factors was not cooperative. Thus, the frequently observed synergy between IFNgamma and TNFalpha in promoting inflammatory response depends in part upon cooperation between STAT1alpha and NF-kappaB, which is most likely mediated by their independent interaction with one or more components of the basal transcription complex.

The cellular and molecular mechanisms of IL-12-mediated anti-tumor activity have been examined. BALB/c mice bearing established s.c. RENCA or CT26 tumors that were treated daily with IL-12 showed essentially complete tumor regression while tumors in untreated animals grew progressively. Examination of inflammatory gene expression in tumor tissue from treated vs untreated mice revealed the selective expression of IFN-gamma and the IFN-gamma-inducible CXC chemokine IP-10. Immunohistologic analysis demonstrated that tumors from treated mice were heavily infiltrated with CD8+ T cells and Mac-1+ mononuclear cells. Tumor regression in IL-12-treated mice was associated with expression of the lytic effector molecules perforin and granzyme B. These findings support the hypothesis that the anti-tumor function of IL-12 treatment depends upon the induced expression of IFN-gamma by T cells and/or NK cells, the amplification of the immune response mediated by IFN-gamma-induced expression of chemoattractant cytokines, and the IL-12-dependent potentiation of the cytolytic effector function of recruited CD8+ T cells.

The present study investigates mechanisms involved in cooperation between IFN-gamma and TNF-alpha to promote transcription from the IP-10 gene in NIH 3T3 cells. IFN-gamma synergistically enhanced TNF-alpha-induced levels of IP-10 mRNA, whereas levels of JE (MCP-1) or KC (GRO/MGSA) mRNA induced by TNF-alpha were unaffected by IFN-gamma. The cooperation between IFN-gamma and TNF-alpha for induction of IP-10 mRNA was independent of de novo protein synthesis and mediated at least in part by increased transcription. Transient transfection analysis with a 243-bp fragment flanking the transcription start site of the murine IP-10 gene indicated that synergy between the two stimuli was dependent upon occupancy of at least two of three critical regulatory sequence elements: an IFN-stimulated response element (ISRE) and one of two kappa B sites. IFN-gamma and TNF-alpha independently activated nuclear factors capable of specific interaction with the ISRE and kappa B sites, respectively. IFN-gamma induced two ISRE binding complexes, one of which was protein synthesis independent, appeared within 15 min of stimulation, and contained p91 or signal transducer and activator of transcription (STAT) 1. TNF-alpha induced only one ISRE binding activity, which was dependent upon protein synthesis. TNF-alpha also induced kappa B binding activity that was composed of NF-kappa B1 (p50) and RelA (p65) whereas IFN-gamma had no detectable effect on kappa B binding activity. Together these results indicate that the highly synergistic transcriptional activation of the IP-10 gene by IFN-gamma and TNF-alpha involves the cooperation between factors that are independently activated by the two stimuli and that bind to independent sites.

IFN-gamma and LPS have both been shown to stimulate enhanced chemoattractant cytokine gene expression in mononuclear phagocytes. In this report, IFN-gamma was found to suppress LPS-induced chemokine mRNA expression in a cell type- and gene-specific fashion. Expression of JE (monocyte chemoattractant protein-1) and KC (GRO/melanoma growth-stimulating activity) mRNA in macrophages stimulated with LPS was markedly suppressed by IFN-gamma in a dose- and time-dependent fashion. LPS-induced IP-10 mRNA was unaffected by IFN-gamma under identical experimental conditions. This effect was cell type-specific because JE and KC mRNA expression in LPS-stimulated murine endothelial cells, TNF-alpha-stimulated endothelial cells, and NIH-3T3 cells were unaffected by IFN-gamma. The IFN-gamma-mediated suppression of LPS-stimulated KC mRNA expression was independent of protein synthesis and mediated at the transcriptional level. These observations indicate that IFN-gamma may function as a negative regulatory signal for the expression of some proinflammatory cytokines in macrophages. The cell type-dependent differential behavior of individual members of the chemokine family may be an important determinant of the cellular composition and outcome of an inflammatory response.

The ability of T cell-derived cytokines to induce the expression of the nitric oxide synthase (NOS) gene in murine peritoneal macrophages was examined. IL-2 or TNF-alpha alone had no effect either on gene expression or enzyme activity, whereas IFN-gamma had only modest activity. When IL-2 or TNF-alpha were used in combination with IFN-gamma, there was a marked cooperative induction of both mRNA and enzyme activity. The cooperative effects were truly synergistic, as the consequences of combined cytokine treatment were many times greater than was seen with any of the agents acting independently. The expression of NOS mRNA and enzyme activity in response to combined lymphokine treatments was a continuous process reaching optimal levels between 24 and 48 h after stimulation. Concentration dependency for both IL-2 and TNF-alpha suggested that their effects were mediated through interaction with the corresponding defined cell surface receptors. Human rTNF-alpha was as effective a stimulus as murine TNF-alpha; because human TNF-alpha is recognized only by the p55 Type II TNF receptor, this structure appears to mediate the response to TNF-alpha. When IL-2 and TNF-alpha were added at saturating doses in the presence of IFN-gamma, there was an additive effect on NOS mRNA expression suggesting that IL-2 and TNF-alpha cooperate with IFN-gamma through at least partially distinct intracellular signaling pathways. Expression of NOS mRNA in response to IFN-gamma/IL-2 or IFN-gamma/TNF-alpha treatment required protein synthesis, suggesting that cooperative cytokine induction of NOS involves the intermediate expression of new gene products. Such molecular controls for regulation of inducible macrophage gene expression can be contrasted with regulatory control of other inflammatory genes such as IP-10 and TNF-alpha.

The transcriptional regulation of the murine IP-10 gene in lipopolysaccharide (LPS) or interferon gamma (IFN gamma)-treated macrophages was investigated by analysis of regions of the gene that flank the transcription start site. A series of sequence fragments were placed 5' to the chloramphenicol acetyltransferase (CAT) reporter gene and ability to mediate transcription of CAT in response to IFN gamma or LPS treatment was studied following transient transfection in the macrophage-like cell line RAW 264.7. Analysis of larger constructs identified a potential negative regulatory site for IFN gamma response in the region between nucleotide positions -2002 and -930 and a positive regulator for LPS response in the region between bases -930 and -676. A 227-base fragment spanning positions -228 to -2 was the minimal sequence able to mediate LPS- and IFN gamma-dependent transcription of CAT. Deletion of 24 bases, which included a highly conserved IFN stimulus response element (ISRE) from the -228 construct, abolished response to IFN gamma. A 33-base fragment containing the IP-10 ISRE was able to confer both IFN gamma and LPS sensitivity upon a heterologous promoter. The ability of LPS to stimulate CAT via the ISRE was apparently mediated by intermediate expression of endogenous IFN alpha/beta. Elimination of bases -204 to -102 abolished sensitivity to LPS. This region contains two kappa B binding sites. Site-directed mutagenesis of key nucleotides in the ISRE and the two kappa B sites demonstrated that optimal response to IFN gamma required both the ISRE and one of the two kappa B sites, whereas optimal response to LPS required either both kappa B sites or one kappa B site and the ISRE. IFN gamma or LPS treatment induced sequence-specific binding activity for the ISRE and the two kappa B sites. These results indicate that the 230 nucleotides upstream from the transcription start site are important for transcriptional control of the IP-10 gene in response to IFN gamma and LPS. The three defined regulatory elements function in distinct fashion for each of the two stimuli; optimal response to either IFN gamma or LPS requires cooperation between at least two sites.

IP-10 is an early gene induced in multiple cell types by a variety of proinflammatory agents, notably interferons (IFNs) and lipopolysaccharide (LPS). To determine whether this protein might play a role in amplifying immune-mediated glomerular injury, we cultured mouse mesangial cells with several stimuli for various times. Increasing amounts of IFN-gamma (to 100 units/ml) elicited increasing levels of IP-10 messenger RNA (mRNA), sustained to 24 hours, but had no effect on tumor necrosis factor-alpha (TNF-alpha) mRNA. LPS induced transient IP-10 mRNA expression that peaked at 8 hours; TNF-alpha mRNA was also increased. TNF-alpha at doses up to 10 ng/ml and soluble immune complexes up to 150 micrograms/ml antibody evoked 3- to 5-fold increases in IP-10 mRNA expression, much less than the 30- to 70-fold increases seen with IFN-gamma and LPS. We conclude that IFN-gamma, LPS, and other agonists can amplify glomerular immune injury, perhaps via elevated expression of IP-10.

The combination of IFN-gamma and IL-2 has been demonstrated to induce or promote transcription from selected inflammatory cytokine genes in both monocytes and macrophages. In the present report, we have evaluated the ability of these agents to activate nuclear proteins, in murine peritoneal macrophages, that are capable of specific binding to the kappa B nucleotide consensus sequence (e.g., nuclear factor binding the kappa B consensus sequence (NF kappa B)), which is present in the region 5' of the transcription start site of many cytokine genes. Both IFN-gamma and IL-2 treatments alone caused some activation of NF kappa B, as evidenced by the appearance of a characteristic DNA-protein complex in electrophoretic mobility shift assays; in combination, the two agents cooperated to increase binding activity markedly, to a level equivalent to that seen in macrophages stimulated with LPS, another potent stimulus of macrophage cytokine gene expression. The binding activity was directed toward the kappa B sequence, based upon competition studies with several different oligonucleotide probes containing the kappa B motif in different contexts. The complex formed in cells stimulated with either IFN-gamma/IL-2 or LPS contained two proteins, of approximately 50 kDa and 65 kDa, based upon UV cross-linking experiments with a bromodeoxyuridine-substituted oligonucleotide probe. Although the effects of LPS and IFN-gamma/IL-2 on NF kappa B activity were both transient, response to LPS was detected earlier and declined more rapidly than that seen with IFN-gamma/IL-2 treatment. The cooperative activation of NF kappa B was dose dependent for both IFN-gamma and IL-2. The activation of NF kappa B by IFN-gamma/IL-2 did not depend upon protein synthesis. These results suggest that the induction of cytokine gene expression in macrophages by the combination of IFN-gamma and IL-2 may involve the activation of NF kappa B.

The effect of IL-4 on inflammatory gene expression in murine peritoneal macrophages stimulated with IFN-gamma in combination with IL-2 has been examined. These agents cooperatively induce the expression of mRNA for TNF-alpha and IP-10. Murine rIL-4 suppresses cytokine mRNA expression depending on the stimulus used and the mRNA being measured. Expression of TNF-alpha mRNA in macrophages stimulated with IFN-gamma, IL-2, or the combination is markedly suppressed by IL-4 whereas LPS-induced TNF-alpha mRNA is unaffected. In contrast, IP-10 mRNA expression is more sensitive to suppression by IL-4 when stimulated by LPS than by IFN-gamma/IL-2. IL-4-mediated suppression does not alter the time course of mRNA expression. Treatment of IFN-gamma/IL-2-stimulated macrophages with cycloheximide blocks the suppressive effect of IL-4, suggesting that de novo synthesis of an intermediate protein is part of the suppressive mechanism. The IL-4-mediated suppression of IFN-gamma/IL-2-driven TNF-alpha gene expression appears to be mediated at the level of transcription. These findings support a role for IL-4 as an antiinflammatory cytokine and suggest that macrophage inflammatory function will be dependent on the precise stimulus composition of the tissue microenvironment.

We have examined the effects of interleukin 2 (IL-2) treatment either alone or in combination with interferon (IFN) gamma or beta on the expression of several activation associated genes (e.g. tumor necrosis factor alpha (TNF alpha), IFN-inducible 10-kDa protein (IP-10] in murine peritoneal macrophages. IL-2 alone did not induce the expression of either gene while IFN gamma or IFN beta had a modest inductive effect on IP-10 but no effect on TNF alpha expression. When either form of IFN was used in combination with IL-2 there was a marked synergistic induction of both mRNAs. IFN gamma and IL-2 were maximally effective when both agents were added simultaneously, and induced mRNA expression declined over a 24-h period in cells pretreated with IFN gamma prior to addition of IL-2. Expression of both genes following combination lymphokine treatment was mediated by increased transcriptional activity. The responses to all three lymphokines were dose dependent; the concentration requirement for IL-2 indicated interaction with an intermediate or low affinity receptor. The expression of both monokine genes was transient and was comparable although not identical to that seen in cells stimulated with lipopolysaccharide. IFN gamma and IL-2 could synergize to stimulate the expression of either TNF alpha or IP-10 mRNA using sequential non-overlapping treatment periods regardless of the order in which the two stimuli were applied to the cells. The effect of either agent alone induced a transient (1-5 h) responsive state with respect to subsequent stimulation with the second agent. Expression of both monokine genes in response to IFN gamma/IL-2 treatment was independent of protein synthesis as cycloheximide did not inhibit the accumulation of specific mRNA. Interestingly, the combination of IL-2 and cycloheximide was as effective as IFN gamma and IL-2 together. In concert, these results indicate that both IFN and IL-2 generate independent intracellular signals which alone are incapable of inductive effect but which are potent activators of inflammatory gene expression when coincidentally expressed.

Macrophage activation for tumoricidal and microbicidal functions can be achieved in part by treatment with recombinant interferon gamma (IFN gamma) in vitro. We have previously demonstrated that IFN gamma treatment of murine peritoneal macrophages results in a two- to five-fold increase in the activity of Ca++, phospholipid dependent protein kinase C (Hamilton et al., J. Biol. Chem., 260:1378, 1985). We now report that this effect was not dependent upon continuing protein synthesis since treatment with cycloheximide under conditions where normal protein synthesis was inhibited by greater than 95% had no effect upon the development of increased enzyme activity. Examination of Ca++ and phospholipid requirements revealed no differences between enzyme isolated from control or IFN gamma treated cells. Similarly, protein kinase C from control and IFN gamma-treated cells could not be distinguished in terms of the diacylglycerol (DG) or phorbol diester (PMA) concentration required for stimulation of activity. Kinetic analysis of the ATP (as substrate) concentration dependence revealed that both control and treated enzyme preparations (either basal or stimulated) had comparable Km values. Maximum velocity (Vmax) was increased both by IFN gamma treatment and also by stimulation with DG or PMA. The major difference which could be discerned between protein kinase C derived from control versus IFN gamma-treated macrophages was the magnitude of the response to DG or PMA; IFN gamma treatment increased the stimulation index (i.e., ratio of basal to stimulated activity) by a factor of two to four fold. These results suggest that IFN gamma treatment leads to reversible modulation of existing protein kinase C resulting in increased catalytic efficiency when exposed to an appropriate stimulant.

The expression of transferrin receptors on murine peritoneal macrophages has been shown to be down regulated during functional activation in vivo. This observation suggested that the level of transferrin receptor expression varies in response to discrete extracellular signals known to induce macrophage activation. We have tested this concept directly and have shown that decreased transferrin receptor expression can be reproduced in vitro by treatment of inflammatory macrophages with preparations of interferon gamma derived from a T cell hybridoma supernatant. The ability of this agent to down regulate the expression of the transferrin receptor exhibited dose and time dependencies similar to those required for development of other macrophage functions in vitro. The addition of LPS produced no further decrease in receptor expression. Furthermore, murine gamma interferon, produced by recombinant DNA technology also caused a downshift in transferrin receptor expression at doses similar to those which have been shown previously to induce activation. The changes in receptor activity were the result of altered numbers of binding sites and the receptor:ligand affinity remained unaffected. These results indicate that altered expression of the transferrin receptor is one element of the pleiotypic change which macrophages undergo in response to IFN gamma. This system may, therefore, provide a useful model in which to study the biochemical basis of IFN gamma action in mononuclear phagocytes.

The number of transferrin receptors in thioglycollate-elicited murine peritoneal macrophages is markedly depressed after exposure to murine gamma-interferon (IFN gamma) in vitro. This change has been used as a model system to study the molecular and cellular mechanisms of IFN gamma signal transduction. We observed that the downshift of the transferrin receptor could be mimicked by exposure to the calcium ionophore (A23187) or the potent tumor promoter, phorbol 12-myristate 13-acetate (PMA). Saturation binding studies on thioglycollate (TG)-elicited peritoneal macrophages after exposure to A23187 or PMA showed the reduced expression of transferrin binding activity attributable to a decrease in the total number of cellular transferrin receptors and not an alteration in receptor-ligand affinity, in agreement with previous results obtained after exposure to IFN gamma. The loss of transferrin receptors in response to A23187 or PMA was dose dependent, and the kinetics of the change were identical to those observed with IFN gamma treatment. Phorbol 12,13-dibutyrate or 4-beta-phorbol 12,13-didecanoate, both biologically active phorbol esters, also induced reduced expression of transferrin receptors, whereas nonesterified phorbol or 4-alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, had no effect on transferrin receptor expression. Finally, PMA and A23187, when used together, acted cooperatively to modulate transferrin receptor expression when both agents were present at subthreshold concentrations. These results, taken together, suggest that elevation of intracellular Ca++ levels and/or stimulation of protein kinase C are involved in the response of macrophages to IFN gamma.

Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.

IFN-gamma can induce the expression of both class II histocompatibility antigens (Ia) and the lymphocyte function associated (LFA)-1 antigen on murine peritoneal macrophages. We have examined the molecular changes which lead to altered expression of these two cell surface proteins to determine whether they are regulated by similar or independent mechanisms. While I-A antigen expression can be induced or enhanced by treatment of macrophages with either phorbol diesters and/or the Ca2+ ionophore A23187, these agents had no effect upon expression of LFA-1 under similar conditions. Macrophages from the A/J strain mouse exhibit a deficiency in their sensitivity to IFN-gamma which is seen in our studies as an inability of IFN-gamma to elevate I-A antigen expression. However, expression of I-A could be modulated in these cells by treatment with either phorbol diesters or A23187. In contrast, IFN-gamma could induce LFA-1 antigen on A/J derived macrophages and this was not affected by either phorbol or A23187. Thus these two antigens, despite coordinate expression in response to IFN-gamma in normal mouse strains, are clearly regulated independently. These results suggest that IFN-gamma generates at least two independent molecular events in macrophages which ultimately modulate the expression of cell surface proteins important to the performance of activated functions.

The content of Ca2+-, phospholipid-dependent protein kinase activity (protein kinase C) in murine peritoneal macrophages treated with recombinant interferon-gamma (IFN-gamma) has been investigated. Protein kinase C activity was solubilized by nonionic detergent extraction of sonicated cells and separated by high performance liquid chromatography on a TSK 4000 SW gel filtration column. The enzyme eluted from the column in a molecular weight range of 60-80 X 10(3) and was identified by virtue of Ca2+ and phospholipid requirements. Macrophages treated with recombinant IFN-gamma exhibited a substantial increase in total protein kinase activity which could be accounted for entirely by increased protein kinase C activity. This activity was enhanced as much as 5-fold over that seen in untreated macrophages and was specific for IFN-gamma in that other agents known to signal changes in macrophage function had no effect. The time required for the elevation of kinase activity was identical to that required for induction of other functions by IFN-gamma in macrophages. These observations suggest that protein kinase C may be a focus of regulatory action in IFN-gamma-mediated macrophage activation.

The functional and biochemical responses of macrophages derived from the A/J mouse strain to IFN-gamma have been studied. As compared to macrophages obtained from C57BL/6 strain mice, cells from mice of the A/J strain are deficient in their response to IFN-gamma for acquisition of tumoricidal competence. This deficiency was not due to reduced expression of surface receptors for IFN-gamma or to altered affinity of the receptor for its ligand. IFN-gamma recently has been shown to enhance the potential activity of protein kinase C (PKc) and to modulate the efflux of intracellular Ca2+ in macrophages from C57BL/6 mice. Neither of these two biochemical changes were induced in macrophages derived from A/J mice. Functional competence could, however, be pharmacologically induced in both C57BL/6- and A/J-derived macrophages by combined treatment with an ionophore plus phorbol myristic acetate, which increase intracellular Ca2+ and stimulate PKc, respectively. Although the exact nature of the deficit in A/J strain mice has not been defined, the present findings indicate that it lies between the expression of receptor and the modulation of PKc activity and Ca2+ levels. Furthermore, the data provide support for the notion that these molecular changes are important components of the stimulus-response coupling process in IFN-gamma-mediated activation of macrophages.

The secretion of tumor necrosis factor (TNF) by macrophages is initiated by lipopolysaccharide (LPS); considerable evidence indicates that such secretion can be potentiated by interferon-gamma (IFN-gamma). The present studies show that accumulation of mRNA for tumor necrosis factor, which represents an important regulatory focus for controlling secretion of TNF, is enhanced by physiologic doses of IFN-gamma (20 units/ml of purified recombinant IFN-gamma). mRNA for TNF induced by LPS, which was maximal 2 hr after LPS was applied to the cells, was enhanced 5- to 8-fold by IFN-gamma as determined by Northern blot analysis. Interferon did not change the kinetics of accumulation but did change the dose effects of LPS in that increasing amounts of LPS led to increasing amounts of TNF mRNA in IFN-gamma-treated macrophages. IFN-gamma itself, however, did not induce expression of TNF mRNA. These studies document that IFN-gamma potentiates the cytoplasmic accumulation of mRNA for TNF induced in murine peritoneal macrophages by LPS.

We have recently described the isolation and characterization of a set of cDNA encoding genes whose expression is induced or enhanced in murine peritoneal macrophages by treatment with LPS. In the present report we have analyzed the expression of the mRNA which hybridize with these cDNA probes in macrophages treated with other cytokines known to modulate functional activity. Three distinct patterns of expression have been documented. Two genes (D3 and C7) are inducible by LPS, IFN-gamma, and IFN-beta; D3 is comparably sensitive to all three, whereas C7 is more sensitive to LPS and IFN-gamma than to IFN-beta. The mRNA encoded by D8 is expressed in response to LPS and IFN-beta but not in response to IFN-gamma. Finally the gene encoded by D5 is inducible only in cells treated with LPS. The expression of all three cytokine-inducible mRNA was both dose and time dependent and was mediated by increased transcriptional activity of the genes. As with stimulation by LPS, the expression induced by IFN was independent of protein synthesis and occurred in a rapid and transient fashion. TNF-alpha had little or no detectable effect on any of the genes by themselves. The expression of C7, however, could be induced synergistically by treatment with a combination of TNF-alpha and either IFN-gamma of IFN-beta. The expression of these genes was not specific for macrophages as both IFN were able to induce a comparable pattern of gene expression in BALB/c 3T3 cells. Treatment of macrophages with dexamethasone inhibited LPS-induced C7 and D8 expression but did not affect that seen in response to IFN-gamma or IFN-beta, respectively. The results suggest that IFN and LPS act to modulate early gene expression by the generation of at least three overlapping but distinct signaling pathways. In some cases the pathway(s) which mediate response to LPS appear to be mechanistically distinct from those which mediate response to IFN-beta or IFN-gamma. The spectrum of stimuli and cell types which express these and other early genes suggest that they may play an important role in orchestration of the inflammatory response.

Maleylated bovine serum albumin (maleyl-BSA) and other polyanionic polymers that are recognized by cell surface receptors on macrophages have been shown to induce chemotaxis, protease secretion, and tumoricidal function in this cell type. In this paper the effect of maleyl-BSA on Ia antigen expression has been evaluated. In a fashion similar to LPS, maleyl-BSA suppressed IFN-gamma-induced expression of Ia in a time- and dose-dependent manner. Also like LPS, maleyl-BSA stimulated the production and secretion of substantial amounts of PGE2 over a 24-hr period. This did not, however, appear to be the primary mechanism by which expression of Ia was suppressed, because co-treatment of the cells with indomethacin, which totally inhibited the production of PGE2, only minimally affected the suppressive activity. Surprisingly, the suppressive activity of both maleyl-BSA and LPS could be largely abrogated by co-treatment of the cells with cyclohexamide during the time period when Ia expression was sensitive to suppression. This effect was selective in that PGE2- or dibutyryl cyclic AMP-induced suppression of Ia expression was not affected by cyclohexamide treatment. The data support the concept that there are multiple molecular mechanisms involved in the negative regulation of IFN-gamma-induced Ia expression in macrophages. Such mechanisms may include, in addition to the synthesis of PGE2 and consequent elevation in intracellular levels of cyclic AMP, one or more proteins made early after treatment with either maleyl-BSA or LPS. Thus the function of some of these early gene products may be to regulate expression of functional genes such as that encoding Ia antigen.