SJL and NOD macrophages are uniquely characterized by genetically programmed, elevated expression of the IL-12(p40) gene, suggesting a conserved pathway for the induction of organ-specific autoimmunity.

Genetic susceptibility of the SJL mouse to experimental autoimmune encephalomyelitis (EAE) appears, in part, to be a result of genes that promote abnormal development of the pathogenic Type 1 (Th1) phenotype of neuroantigen-specific T-cells. Because antigen-presenting/accessory cells (APCs) produce cytokines that can modulate the development of Th1 and Th2 phenotypes, we addressed whether APCs from SJL mice were genetically programmed for elevated expression of the Th1-promoting cytokine, IL-12. Activated peritoneal macrophages (Mphi; i.e., APC) from naïve SJL mice produced levels of TNF-alpha, IL-1, IL-6, IL-10, and TGF-beta within the range of six normal strains. In contrast, SJL IL-12p40 (in addition to IL-12p70) production was consistently five- to 20-fold greater than that of any normal strain tested, which arose from elevated expression of the IL-12p40 but not the IL-12p35 gene, because p40 mRNA levels were eight- to 15-fold greater than those of normal strains. This aberrancy in IL-12p40 expression appears identical to that observed in the NOD mouse, another strain prone to organ-specific autoimmunity. A genetically programmed bias toward elevated expression of IL-12 in Mphi from the SJL and NOD strains of autoimmunity provides a conserved mechanism for the dominant Th1 development of naive, autoantigen-specific T-cells in these strains. This study is the first demonstration of a genetically programmed aberrant phenotype that is intrinsically expressed within a cell type in the SJL mouse and provides insight into its predisposition for EAE.

Aberrant macrophage cytokine production is a conserved feature among autoimmune-prone mouse strains: elevated interleukin (IL)-12 and an imbalance in tumor necrosis factor-alpha and IL-10 define a unique cytokine profile in macrophages from young nonobese diabetic mice.

Cytokines derived from macrophages (Mø) play a critical role in the development of type 1 diabetes in the nonobese diabetic (NOD) mouse. Based on earlier findings from lupus-prone strains of inherent cytokine defects in Mø , NOD Mø were evaluated for intrinsically dysregulated cytokine production with the potential to initiate or exacerbate disease. Endotoxin-activated peritoneal Mø from young prediseased NOD mice produced interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha levels similar to those of Mø from a panel of control strains but reduced compared with the congenic diabetes-resistant NOR strain. IL-6 and IL-10 production were similar in NOD and NOR Mø, indicating that reduction in NOD IL-1 and TNF-alpha expression was selective. Nevertheless, the ratio of TNF-alpha and IL-10 production, a stringent index of normal Mø function, distinguished NOD from all normal strains. The most striking feature of NOD Mø, however, was their substantially elevated IL-12 production. This response was induced not only by endotoxin but also by bacillus Calmette-Guerin (BCG) and CD40 ligand and was associated with (and likely caused by) the enhanced and prolonged expression of p40 mRNA. Moreover, NOD Mø IL-12 expression appeared to be near maximally induced by lipopolysaccharide (LPS) alone, because it was only slightly enhanced by the addition of gamma-interferon, a stimulus that substantially elevated LPS-induced IL-12 production in Mø from normal strains. Accompanied by a unique profile of TNF-alpha and IL-10, the dramatic elevation of IL-12 expression by NOD Mø reflects intrinsic defects of the innate immune system with the potential to initiate and propagate the pathogenic autoreactive T-helper type 1 response characteristic of type 1 diabetes.

Intrinsic defects in macrophage IL-12 production associated with immune dysfunction in the MRL/++ and New Zealand Black/White F1 lupus-prone mice and the Leishmania major-susceptible BALB/c strain.

We have demonstrated that macrophages (Mphi) from young, prediseased, lupus-prone MRL/++ and New Zealand Black/White F1 mice display defective production of TNF-alpha, IL-1, and IL-6, but normal production of IL-10. In an attempt to determine the potential functional implications of this phenotype for autoimmunity, we demonstrate here that endotoxin-activated Mphi from these lupus-prone mice showed dramatically reduced expression of IL-12, a cytokine essential for Th1 responses that may be defective during lupus. IL-12 production was also reduced by Mphi from the control BALB/c strain, compatible with the concept that a genetically programmed deficit in IL-12 levels may underlie the IL-4-dominated BALB/c response to infection by the parasite Leishmania major. Although both IL-12 and TNF-alpha expression defects by Mphi from lupus-prone strains are expressed rapidly after activation, treatment with each cytokine demonstrated that only TNF-alpha contributes to the subsequent dysregulation of Mphi IL-1 and IL-6 expression in these strains, and that the reduced autocrine activity of defective IL-12 or TNF-alpha levels was not causal to each other. Although the intrinsic defect in IL-12 expression by lupus-prone and BALB/c Mphi may lead to defective Th1 responses, these Mphi responded to the Th1-derived cytokine, IFN-gamma, in a normal fashion suggesting a defective role in the induction, rather than the propagation, of Th1 responses in these mice. Our finding of a conserved intrinsic defect in IL-12 production by Mphi from the two principal mouse models of multigenic lupus provides insight into how excessive humoral responses may develop, and perhaps be prevented, in systemic autoimmune disease.

Adhesion elicits an intrinsic defect in interleukin-1 expression by macrophages from autoimmune-prone MRL mice.

Macrophages (m phi) from prediseased autoimmune-prone MRL/+ and MRL/ lpr mice have a marked defect in endotoxin (LPS)-induced expression of several cytokines including interleukin 1 (IL-1). The progressive nature of this defect over time suggests that it may develop in response to specific extracellular stimuli. In this report, we show that adhesion is an essential factor for the development of aberrant IL-1 expression by m phi from autoimmune-prone MRL mice. Thus, when MRL/+ m phi were allowed to adhere before being stimulated with LPS, they demonstrated a striking defect in expression of both IL-1 message and protein in comparison with multiple normal strains. In marked contrast, when MRL/+ m phi were maintained in a non-adherent state by culture on agarose, the IL-1 defect was not evident and IL-1 expression was restored to nearly normal levels. Since an identical defect in IL-1 expression was found when MRL/+ m phi were cultured on a variety of extracellular matrix proteins (including laminin, fibronectin, type I collagen, and type IV collagen), it appears that IL-1 underexpression is dependent on the adhesive state per se rather than on engagement of any one specific adhesion receptor. Moreover, the cytoskeletal inhibitor cytochalasin D had no effect on the magnitude of the defect, indicating that the adhesion-dependent signaling events necessary to elicit IL-1 underexpression are independent of cytoskeletal rearrangement. Taken together, these results indicate that m phi from autoimmune prone MRL/+ mice have an adhesion-dependent signaling abnormality that leads to profound underexpression of the cytokine IL-1.

IL-15 functions as a potent autocrine regulator of macrophage proinflammatory cytokine production: evidence for differential receptor subunit utilization associated with stimulation or inhibition.

The cytokine IL-15 appears to mimic the stimulatory activity of IL-2 on lymphocytes by utilizing part of the IL-2R complex. Although effects of IL-15 on Mphi activities have not previously been reported, its derivation from activated Mphi suggested a possible autocrine role in regulating Mphi functions and prompted us to determine whether IL-15 modulated LPS-activated Mphi cytokine production. Whereas high IL-15 concentrations enhanced proinflammatory (i.e., TNF-alpha, IL-1, and IL-6) and anti-inflammatory (i.e., IL-10) cytokine production by two- to sixfold, extremely low IL-15 concentrations (picomolar to attomolar range) markedly and selectively suppressed Mphi proinflammatory, but not anti-inflammatory, cytokine production by two- to fourfold. The stimulation (but not the suppression) of TNF-alpha production by IL-15 required the (IL-2/IL-15) receptor beta chain, as demonstrated by receptor subunit-blocking studies and lack of stimulation of Mphi from IL-2Rbeta-deficient mice. Conversely, suppression most likely involved the alpha receptor (IL-15R alpha) because this high affinity receptor would be engaged by low concentrations of IL-15, and its inducible expression correlated with the degree of suppression in both a time- and LPS dose-dependent fashion. Moreover, Ab-mediated neutralization studies revealed that endogenous IL-15 activity regulated Mphi activation with kinetics similar to that seen in response to exogenously added IL-15: suppressor activity increased over time in correlation with IL-15R alpha gene expression. This study demonstrates a novel dose-dependent and autocrine activity of IL-15 in Mphi regulation.

Aberrant cytokine expression and autocrine regulation characterize macrophages from young MRL+/+ and NZB/W F1 lupus-prone mice.

We investigated whether macrophages (Mphi) from young, lupus-prone MRL+/+ and NZB/W F1 mice expressed common defects in immunoregulatory cytokine production. Endotoxin-activated Mphi from both strains, obtained well before disease signs, had a markedly reduced capacity to maintain IL-1 production compared with Mphi from normal strains (BALB/c, A/J, and C57BL/6). Mphi from lupus-prone mice showed similar defects in IL-6 and TNF-alpha production, which preceded the IL-1 defect. In fact, defective TNF-alpha production appeared to be responsible for aberrant expression of the other cytokines because this defect was the first to be expressed, and treatment with exogenous TNF-alpha reduced the extent of defective IL-1 and IL-6. These "proinflammatory" cytokine defects appeared to be selective because the anti-inflammatory cytokine IL-10 was not expressed aberrantly in the lupus-prone strains. For this reason, and because anti-IL-10 mAb treatment did not correct defective proinflammatory cytokine production, IL-10 did not appear to be responsible for these defects. IFN-gamma was able to normalize TNF-alpha production in Mphi from lupus-prone mice, demonstrating a stimulus-specific induction of the proinflammatory defects. These studies also revealed that Mphi from the three normal strains studied here maintain a precise inverse relationship between levels of TNF-alpha and IL-10, a relationship not seen in Mphi from lupus-prone strains. These findings reveal shared elements of cytokine dysregulation in the two principal animal models of multigenic lupus, and suggest that the study of Mphi (and perhaps other cells of the innate immune system) may provide valuable insights into intrinsic functional defects associated with systemic autoimmunity.

Endogenous substance P mediates cold water stress-induced increase in interleukin-6 secretion from peritoneal macrophages.

Previous studies from this laboratory had shown that exposure of mice to cold water stress leads to an increase in the secretion of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF alpha) from their peritoneal macrophages. We now report that the secretion of IL-6 from peritoneal macrophages is also increased after cold water stress and that the peptide substance P (SP) participates in this stress-induced response. The stress paradigm involved subjecting male C57BL/6J mice to 5 min swim tests in 10 +/- 2 degrees C water twice daily for 4 d. Cold water stress augments the lipopolysaccharide-induced IL-6 secretion from peritoneal macrophages, elevates immunoreactive SP (iSP) in the peritoneal wash fluid, and reduces iSP in certain peritoneum-containing tissues or organs (i.e., diaphragm, abdominal wall, ileum, and rectum). The 10 d stress time studies indicate that increased IL-6 secretion is positively related to elevated iSP in the peritoneal wash fluid and inversely related to reduced iSP in certain peritoneum-containing tissues. Pretreatment with capsaicin, which depletes SP in the sensory nerve endings, eliminates stress-control differences in the peritoneal wash fluid and in certain peritoneal tissues. Moreover, RP67,580, a specific SP antagonist, eliminates the cold water stress-induced augmentation of IL-6 secretion from peritoneal macrophages. These results suggest that cold water stress promotes the release of SP from peritoneal tissues into the peritoneal cavity, where it participates in the cold water stress-induced macrophage functional alterations.

Dysregulated cytokine expression in vivo in prediseased and diseased autoimmune-prone MRL mice.

Macrophages (mø) from prediseased autoimmune-prone MRL/ + and MRL/lpr mice produce markedly decreased levels of IL-1 in vitro in response to LPS. In contrast, tissues from diseased MRL/lpr mice overexpress IL-1 in vivo. To determine whether IL-1 underproduction in the MRL strains is solely an in vitro phenomenon, we compared in vivo cytokine mRNA expression from prediseased age-matched MRL/ + and MRL/lpr mice to that from normal BALB/c and C3HeB/FeJ mice. Like mø in vitro, whole organ RNA from the spleen, liver, and kidney of MRL/ + and MRL/lpr mice showed down-regulation of IL-1 RNA following intraperitoneal injection of LPS. This abnormality in inducible IL-1 expression was present in all MRL mice, irrespective of disease stage or the presence of the lpr gene. On the other hand, only diseased MRL/lpr mice displayed elevated and constitutive expression of IL-1 in their livers and kidneys. We suggest that inducible expression is most indicative of the intrinsic, or genetic, capacity of cells to produce cytokine, whereas constitutive expression reflects extracellular disease-related inflammatory stimuli present only in the diseased MRL/lpr strains. By restricting our studies to prediseased MRL mice, we have tried to eliminate the effects of disease and to focus on the predisposing genetic background. The existence both in vitro and in vivo of a defect in inducible IL-1 expression by prediseased MRL mice suggests that the molecular abnormality underlying this defect may be a part of this predisposing background to autoimmunity.

Substance P and stress-induced changes in macrophages.

The present paper further links nervous-endocrine-immune systems by describing influences of SP on the immune system, and more specifically, on macrophage function. We have discussed how macrophages are important to immune responses in that much of cellular and humoral responses depend on macrophage function. Macrophages are sensitive to stress in that cold-water stress causes increased cytokine production, either spontaneously (IL-1), or after induction with LPS (IL-6, TNF alpha). Increased cytokine levels (IL-1, IL-6) may induce acute phase reactants in the liver, which is presumably the mechanism operative in the studies indicating increases in acute phase reactants after certain stressors in animals. SP is a likely candidate to affect immune function. Previous data show that macrophages from various species have receptors for and respond to SP in vitro. SP stimulates phagocytic and chemotactic capacity, as well as increased cytokine, PGE2, and thromboxane B2 production. SP is also involved in neurogenic inflammation and is likely to be involved in the pathogenesis of several inflammatory diseases. Present data indicate SP's involvement in macrophage responses to stress. We have shown that stress induced differential SP receptor binding to peritoneal macrophages, although the precise nature of binding differences has not yet been clearly elucidated. Stress also induces more immunoreactive SP in the peritoneal fluid that bathes the peritoneal macrophages. We hypothesize that the two events, altered SP binding and concomitant increased ligand, are causally related. In addition to other correlational data showing concomitant increased SP binding plus ligand concentrations, there is more direct evidence that SP ligand may induce SP receptor expression since the SP antagonist, CP-96,345, prevents the induction of SP receptor mRNA in the staphylococcal toxin A-induced gastroenteritis (C. Pothoulakis and S. E. Leeman, personal communication). Further supporting our notion for a causal relationship we have found the elimination of SP in vivo (via capsaicin pretreatment) reduced SP binding, as has been previously reported. We have also examined the role of SP on stress-induced altered macrophage function in vitro. SP greatly enhanced the LPS-induced macrophage TNF alpha production from stressed animals; in contrast, it produced relatively little effect on macrophages from control animals. Capsaicin pretreatment diminished the enhanced cytokine production in response to stress, such that levels of TNF alpha and IL-6 approximated those of control mice. Taken together, past and present data suggest that (1) stress may initiate, or at least contribute to, an inflammatory response, and that (2) SP is involved in the macrophage stress response. SP has long been known to be involved in inflammatory processes; our data further suggest its role in mediating stress-induced cytokine alterations.

Aberrant cytokine regulation in macrophages from young autoimmune-prone mice: evidence that the intrinsic defect in MRL macrophage IL-1 expression is transcriptionally controlled.

We have reported that, when compared to macrophages from normal strains, macrophages from the autoimmune-prone MRL and NZB mouse strains demonstrate dramatically reduced IL-1 expression in response to LPS. In MRL mice, this is an intrinsic defect which is unmodified by age, the progression of disease, or the presence of the Ipr gene. Here we report that the key events leading to aberrant IL-1 expression appear to be transcriptional, based on the following three sets of findings. (1) Nuclear run-on analysis demonstrates that the patterns of IL-1 transcription in MRL/+ and BALB/c macrophages are distinct, as the former is clearly more transient. The reduction in MRL/+ IL-1 transcription coincides with a reduction in the levels of nuclear NF-KB and precedes a drop in IL-1 mRNA steady-state levels. (2) Reduced levels of IL-1 transcripts are found in both nuclear and cytosolic fractions of MRL/+ macrophages, arguing against faculty IL-1 mRNA transport into the cytosol as a contributing factor in the establishment of this defect. (3) In the presence of actinomycin D, the rate of RNA degradation is similar in MRL/+ and BALB/c macrophages. Moreover, in vitro RNA decay assays demonstrate that even in the absence of metabolic inhibitors, there is no evidence for an accelerated decay of IL-1 mRNA during exposure to lysates isolated from MRL/+ vs BALB/c macrophages. Taken together, these findings argue that transcription is the predominant level at which this striking example of cytokine dysregulation is controlled.

Interleukin-1 dysregulation is an intrinsic defect in macrophages from MRL autoimmune-prone mice.

Macrophages (M phi) from pre-diseased autoimmune-prone MRL mice (both MRL/+ and MRL/lpr) dramatically underproduce the cytokine interleukin-1 (IL-1) in comparison to M phi from a number of normal strains. In this study we show that IL-1 dysregulation by MRL M phi is fully expressed at birth, and that this defect does not change with time or the development of disease. We also constructed adult irradiation chimeras (consisting of A/J-->MRL and MRL-->A/J mice), and show that M phi isolated from these chimeras display a pattern of IL-1 production indistinguishable from that of the donor strain controls. Moreover, when we constructed a mixed chimera (A/J + MRL-->A/J, the A/J and MRL M phi coexisting within the same animal retained their individual patterns of IL-1 production when isolated by negative selection. Taken together, these results provide the first substantive evidence for an intrinsic defect (IL-1 dysregulation) in M phi from MRL autoimmune-prone mice.

Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes.

Monocytes play a critical role in defending the host against foreign organisms and in regulating the behavior of other cells. Monocytes circulate as nonadherent cells in the blood and migrate as adherent cells through tissues. Adhesion molecules mediate not only cell adhesion, but also migration, phagocytosis, and many other adhesion-dependent functions. Monocyte chemoattractant protein-1 (MCP-1) is thought to be responsible for monocyte recruitment in acute inflammatory conditions and may be an important mediator in chronic inflammation. In this study, immunofluorescence flow cytometry was used to determine whether MCP-1 can regulate the cell surface expression of adhesion molecules, particularly beta-2 and alpha-4 integrins and the leukocyte adhesion molecule-1. We found that MCP-1 induced expression of CD11c (p150,95 alpha-subunit) and CD11b (Mac-1 alpha-subunit), and caused little or no change of CD11a (lymphocyte function-associated Ag-1 alpha-subunit), very late activation Ag-4, or leukocyte adhesion molecule-1. We demonstrated that antibodies to beta-2 and alpha-4 integrins inhibited MCP-1-induced monocyte chemotaxis. We also showed that MCP-1 is capable of inducing IL-1 and IL-6, but not TNF production of monocytes. These results indicate that MCP-1 is not only a chemoattractant but also a novel cytokine with the capacity to regulate several parameters of monocyte function.

Imbalanced cytokine production by macrophages from autoimmune-prone mice.

Peritoneal macrophages from multiple autoimmune-prone strains of mice (MRL/lpr, MRL/+, NZB/W, BXSB, and B6/gld) show defective expression of the cytokines IL-1 and IL-6. Autoimmune mice were all used between 1 and 6 weeks of age, the earliest times being well before the onset of overt disease. Northern blot analysis reveals a parallel reduction in the levels of IL-1 alpha, IL-1 beta, and IL-6 mRNA. In contrast, the production of other proteins, including the cytokine TNF-alpha, appears to be normal. These findings imply an imbalance within the cytokine network of autoimmune macrophages. Studies on bone marrow-derived macrophage precursors, as well as macrophages from chimeric mice, suggest an intrinsic macrophage defect as opposed to conditioning by the autoimmune environment. This defect appears to be constant throughout the lifespan of autoimmune MRL/lpr mice, being equally apparent in 1-week old mice as in fully diseased 6-12-month-old mice. Aberrant regulation of IL-1 and IL-6 represents a novel defect in the function of autoimmune macrophages that is both intrinsic and substantial, and has the potential to contribute to the immune dysregulation that characterizes autoimmunity.

Immunosuppression in mice induced by cold water stress.

A number of studies indicate that stress can result in suppression of the immune system in animals and man. Most of the studies have focused on alterations of lymphocyte function while only a few have investigated alterations of macrophage function or macrophage cytokine production. Macrophages play an essential role in homeostasis of the immune response. Indeed, the earliest events of the immune response occur in cells of the monocytic lineage, and their secretion of various cytokines may have both immunological and nonimmunological effects. The present studies were undertaken to determine whether alterations in macrophage physiology occur in mice subjected to a stress stimulus. Our studies in mice exposed to cold water stress for 4 days indicated reduced numbers of thymocytes and splenocytes, decreased T-cell blastogenesis, and reduced NK activity. Examination of elicited peritoneal macrophages from stressed mice revealed increased prostaglandin E2 (PGE2) secretion and decreased immune region associated antigen (Ia) expression in response to interferon-gamma. Despite elevated PGE2 levels, indomethacin was generally unable to restore depressed immune function. Of special interest was the finding that cell-associated and secreted interleukin 1 were significantly higher from unstimulated elicited macrophages from stressed mice. These results suggest that early in the response to stress, functions of a variety of cells within the immune system, especially macrophages, are altered and that dysregulated macrophage function may well contribute to the generalized suppression of the immune response in cold water stressed mice.

Aberrant regulation of IL-1 expression in macrophages from young autoimmune-prone mice.

IL-1 is a multifunctional, immunoregulatory polypeptide produced by many cell types. Because activated macrophages are a major source of IL-1 and have also been implicated in the pathogenesis of autoimmune disease, we investigated the regulation of IL-1 expression in several autoimmune-prone strains of mice. Peritoneal macrophages derived from the autoimmune-prone strains MRL/lpr, MRL/+, NZB, and NZB/W F1, as well as NZW, displayed transient expression of IL-1 in contrast to the stable expression characteristic of control normal strains including A. Thy, A/J, B10, B10.A, B10.D2, C57BL/6, BALB/c, and C3H/HeN. The down-regulation of IL-1 by macrophages from the autoimmune-prone mice was not attributable to inherently defective signal transduction because macrophages from both the normal and autoimmune-prone strains displayed substantial initial levels of cell-associated and secreted IL-1. However, during the first 2 to 3 days in culture, macrophages from autoimmune-prone mice became progressively refractory to both induction and maintenance of IL-1, a pattern that correlated with changes in the levels of IL-1 alpha and beta mRNA. The progressive reduction in IL-1 expression by macrophages from these autoimmune-prone strains was not due to a reduction in general metabolism or viability, because expression of cell surface antigens, including MHC class I and II Ag and LFA-1, was comparable to that of control macrophages. Because IL-1 plays a critical role in the homeostasis of a variety of cell lineages, defective expression, and maintenance of IL-1 (and perhaps other cytokines) by macrophages from the autoimmune-prone strains may contribute to the immune dysregulation that develops in these mice. Alternatively, cytokine dysregulation might not contribute directly to disease, but rather reflect a more basic defect related to specific signal transducing or gene regulatory pathways.

Regulation of macrophage activation by IL-3. II. IL-3 and lipopolysaccharide act synergistically in the regulation of IL-1 expression.

We have previously reported that IL-3, a cytokine produced by both Th1 and Th2 type CD4+ T cells, displays macrophage-activating potential. IL-3, like IFN-gamma, readily induced functions related to Ag presentation (e.g., Ia and lymphocyte function-associated Ag-1 expression). However, in contrast to the response elicited by IFN-gamma, tumor cytotoxicity was not induced by IL-3. In this paper we have evaluated the capacity of IL-3 to regulate IL-1 expression. Our data demonstrate that although IL-3 alone was unable to induce the production of substantial IL-1 bioactivity in peritoneal exudate cells, it contributed synergistically to the induction of IL-1 bioactivity in the presence of suboptimal doses of LPS. It was of interest that IFN-gamma, which can also interact synergistically with LPS, was unable to complement the partial signals provided by IL-3 for the expression of IL-1 bioactivity, suggesting that IL-3 and IFN-gamma may be providing similar stimulatory signals in this respect. Our studies on the mechanism of synergy between IL-3 and LPS indicated that the effect of LPS did not appear to be mediated by the well-characterized LPS-inducible cytokines of macrophage origin (i.e., IL-1, alpha and beta, TNF-alpha, and IL-6). The best characterized function of IL-3 is its multicolony-stimulating activity as a CSF; in this context we also studied granulocyte-macrophage CSF and noted that it behaves similarly to IL-3 in that it can synergistically contribute to IL-1 induction. A similar, but more dramatic induction of IL-1 synthesis in response to IL-3 was demonstrated by the P388.D1 murine macrophage cell line. The kinetics and the molecular mechanism of the response of P388.D1 to IL-3 indicate several unique features of IL-3-induced IL-1 expression: 1) IL-3 itself induced IL-1 mRNA expression, which was unaccompanied by substantial production of bioactivity, either cell-associated or secreted into the culture supernatant; 2) IL-3 synergized with suboptimal doses of LPS to induce not only heightened IL-1 mRNA levels but bioactivity as well; and 3) IL-3, when combined with LPS, altered the kinetics of IL-1 message and bioactive protein production in response to LPS: IL-3 and LPS induced an early release (3 to 7 h poststimulation) of the IL-1 protein as well as a second peak of mRNA and bioactivity (at 12 to 36 h), which was not observed in response to either IL-3 or LPS alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of macrophage activation by IL-3. I. IL-3 functions as a macrophage-activating factor with unique properties, inducing Ia and lymphocyte function-associated antigen-1 but not cytotoxicity.

Here we report that IL-3 (also referred to as multi-CSF because of its colony-stimulating activity on a variety of hemopoietic cell lineages) can function as a macrophage-activating factor (MAF). IL-3 was able to regulate the expression of class II MHC Ag and the cellular interaction molecule lymphocyte function-associated Ag-1 on the surface of murine peritoneal exudate cells. The kinetics of IL-3-induced Ia expression appeared to be distinct from that induced by either IFN-gamma, IL-4, or granulocyte-macrophage-CSF. IL-3 was also distinguished from these factors by the finding that it did not induce macrophage tumoricidal activity. In addition to its inherent MAF activities, IL-3 also showed a marked synergy with low doses of LPS (0.05 to 0.5 ng/ml) as well as IFN-gamma in Ia induction. When lymphocyte function-associated Ag-1 expression was evaluated, the effects of these stimuli appeared to be only additive. Although LPS has been shown to inhibit IFN-gamma-induced Ia expression, in our experiments this property of LPS is manifest only when present at doses greater than or equal to 50 ng/ml. At lower concentrations, LPS potentiated both IL-3- and IFN-gamma-induced class II MHC Ag expression. Data presented here also suggest that the synergistic interactions between low doses of LPS and IL-3 are not mediated by known LPS-inducible cytokines of macrophage origin, because rIL-1, TNF-alpha, or IL-6 did not enhance the response to IL-3. Because IL-3 can also participate in the regulation of IL-1 expression, it appears that IL-3 can function as a MAF which selectively regulates the accessory cell characteristics required for Ag presentation, as opposed to the cytolytic functions of the macrophage.

Induction of macrophage membrane interleukin 1 expression by T-cell dependent and T-cell independent pathways is inhibited by cyclosporin A.

Here we report that cyclosporin A (CsA) inhibits the induction of membrane interleukin 1 (mIL-1) expression on murine peritoneal macrophages. The inhibition of mIL-1 expression was noted in response to both autoreactive T-cell lines specific for class I or class II MHC determinants as well as bacterial endotoxin. The macrophages were the direct target of this inhibition as shown by pretreating T cells and macrophages separately with CsA. The effective suppression by CsA of endotoxin-induced mIL-1 expression was dependent not only on the concentration of endotoxin employed, but also on the relative time of addition of CsA and endotoxin. Furthermore, CsA pretreatment of macrophages abrogated their ability to stimulate synthesis of IL-4 by a Th2 cell clone. These data suggest that inhibition of induction of accessory molecules such as mIL-1 may be a mechanism by which CsA abrogates the capacity of macrophages to present antigen.

Lymphokine-independent induction of macrophage membrane IL-1 by autoreactive T cells recognizing either class I or class II MHC determinants.

Here we report that autoreactive T cell clones and T cell hybridomas that recognize class I or class II MHC determinants can induce IL-1 expression on cultured macrophages in an MHC-restricted manner. This genetic restriction of membrane IL-1 (mIL-1) induction is not absolute, however; it is manifest only in macrophages that have been cultured for several days before stimulation. Macrophages that are evaluated within 24 h after adherence display a basal level of mIL-1, and the T cell-induced augmentation of basal mIL-1 expression is not MHC-restricted. It appears that T cells of both Th1 and Th2 type have the capacity to induce mIL-1, suggesting that this function is not limited to the T cell subset (Th2) that is able to use IL-1. Most importantly, the ability of T cells to induce IL-1 on macrophages seems to occur by virtue of direct cellular interactions, and is independent of lymphokine secretion. The induction event is rapid enough (2 to 4 h) to allow T cells to interact with both antigen and IL-1 during the initial T cell/macrophage contact. These findings thus reveal an efficient mechanism for the induction of IL-1 during Ag presentation to T cells.

Simultaneous expression of Ia and cytocidal activity by macrophages, and the consequences for antigen presentation.

We have investigated whether the interrelationship of Ia expression and cytotoxicity by macrophages, as the two functions if expressed at the same time, might be counterproductive for T-cell development and function. We report that, under some circumstances, there is a clear dissociation of the two activities, as was demonstrated for both in vitro and in vivo conditions. However, the two functions could also be superimposed. Dissociation or superimposition was determined by (i) the nature of the inducing stimulus, and (ii) by the time-span between stimulation and evaluation. It was found that Ia and tumour killing were mainly expressed by the same macrophage population and, as a result, cytolytic activity, when associated with Ia expression, can be directed against T-cell hybridomas in an antigen-specific manner. The physiological relevance of the dissociation or superimposition of Ia expression and tumour killing by macrophages is discussed.