Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor.

Anti-Mac-1 (M1/70), a rat monoclonal antibody that reacts with mouse and human macrophages, polymorphonuclear leukocytes (PMNL), and natural killer cells, selectively inhibited complement receptor-mediated rosetting by murine macrophages and human PMNL. Preincubation of macrophages with anti-Mac-1 inhibited formation of rosettes with sheep erythrocytes bearing IgM antibody and murine C3 fragments. No inhibition was observed when other monoclonal antibodies that react with macrophages (such as anti-Ly5, anti-H-2, or anti-pan-leukocyte) were tested at 10-fold higher concentrations. Anti-Mac-1 did not affect macrophage Fc receptor-mediated rosetting. Erythrocytes bearing homogeneous human C3 fragments C3b (EC3b) or C3bi (EC3bi) were used to test the specificity of the murine macrophage and human PMNL complement receptor inhibited by anti-Mac-1. In both cases, anti-Mac-1 inhibited CR3-mediated rosetting of EC3bi but not CR1-dependent rosetting of EC3b. The results show that Mac-1 is either identical to CR3 or closely associated with CR3 function. This is one of the first cases in which a monoclonal antibody-defined differentiation antigen has been associated with a specific cell surface function.

Demonstration of a soluble mediator that induces exudates rich in Ia-positive macrophages.

Previous studies have shown that Listeria monocytogenes-immune T cells, adoptively transferred into normal mice with killed Listeria organisms, induced peritoneal exudates rich in Ia-positive macrophages. We show now that culture fluids generated by Listeria-immune exudate cells and Listeria contain an activity that elicits Ia-rich exudates when injected intraperitoneally. The factor that recruits Ia-positive macrophages must be injected several times during a 2-d period for optimal demonstration of its activity. The induction of the factor is immunologically specific and requires Ia-positive macrophages, primed T lymphocytes, and antigen challenge. The factor is a nondialyzable protein and is not genetically restricted in its activity. The macrophages in the exudates induced by the factor bear Fc receptors, take up latex, synthesize I-A, but bear few C3 receptors. We have thus identified an immune mediator capable of controlling the Ia phenotype of the exudate macrophages.

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.

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 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.

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.

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.

Functional significance of the regulation of macrophage Ia expression.

The relationship of Ia expression and antigen-presenting function by macrophages has been evaluated. When macrophages are maintained in standard culture media, both Ia antigens and accessory cell function are lost. The reacquisition of these properties follows exposure to an Ia-inducing lymphokine, for which cDNA-derived interferon-gamma may substitute. The induction of function is related quantitatively to the level of Ia expression. Moreover, both properties reflect newly expressed Ia determinants, since treatment with anti-I-A plus complement at the beginning of culture diminishes neither the subsequent level of Ia expression nor function. Treatment with anti-Mac-1 plus complement, however, reduces function commensurate with the effectiveness of macrophage depletion. Finally, we find that fixation of macrophages after exposure to antigen does not inhibit antigen presentation, indicating that metabolic activity, while required for antigen processing, is not necessary for presentation.

Evidence that thymocytes require at least two distinct signals to proliferate.

Optimal receptivity of thymocytes to the mitogenic stimulus in macrophage culture fluid (MCF) is limited to the time in culture when these cells are spontaneously proliferating. By providing a brief, nonmitogenic lectin pulse, nonproliferating cells can be "activated" and their ability to respond to MCF totally restored. This procedure provides a model system for evaluating thymocyte proliferation and, in particular, the mechanism of action of MCF.

IA antigens and antigen-presenting function of thymic macrophages.

Thymic adherent cells were isolated after their enrichment on density gradients. The predominant cell type found was the macrophage, as determined by morphology, surface receptors for Fc and C3, phagocytosis and esterase activity. There was, in addition, a minor fraction of cells with a distinctive dendritic morphology. These dendritic cells had surface properties similar to macrophages but limited phagocytic capacity. Approximately 50% of thymic adherent cells bear Ia antigens detected by immunofluorescence by using either A.TH anti-A.TL or monoclonal anti-I-A antibodies. These cells were also found to be an extremely effective antigen-presenting source for macrophage-depleted immune T cells, supporting the idea that the Ia antigens detected are of functional significance. Our data indicate that the macrophage is the predominant adherent cell type in general, and the principal Ia-bearing cell in particular, isolated by either physical disruption of the thymus or by collagenase dissociation of thymis stroma.

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.

Thymic macrophages modulate one stage of T cell differentiation in vitro.

We have described a procedure for isolating thymic macrophages and have evaluated their activity in stimulating thymocyte maturation in vitro. The culturing of gradient-purified immature thymocytes on thymic macrophages leads to an increased expression of H-2 antigens and decreased lytic sensitivity with anti-TL and C. The macrophage-stimulated thymocyte also acquires the ability to respond in the MLR. We propose that the macrophage may regulate one stage of thymic differentiation in vivo.

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.

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.

The effect of adherence on the in vitro induction of cytocidal activity by macrophages.

The effects of macrophage adherence to plastic on tumoricidal activity were investigated. In order to do so, an agarose culture system was developed to provide a means for maintaining non-adherent macrophages in vitro. We were not able to show any effects of adherence on tumour cell lysis by activated macrophages. On the other hand, in vitro activation of non-adherent macrophages was possible only if adherence was replaced by another triggering signal. This requirement was more significant in the A/J mouse strain, where non-adherent macrophages required longer activation periods. Hence we propose that adherence might provide a second signal for the in vitro induction of tumour killing. The biological significance of adherence that is relevant to tumour killing is discussed.

Regulation of macrophage populations. V. Evaluation of the control of macrophage Ia expression in vitro.

In response to a lymphokine (LK) produced by activated T cells, macrophages can be induced to express Ia in vitro. This appears to be a complex process comprised of a number of discernible events. Peritoneal macrophages elicited by different means, and unstimulated macrophage and monocyte populations, each had a distinct kinetic profile of Ia induction. This response was characterized most noticeably by a latent period before actual Ia expression. The latent period varied from 3 to 7 days, depending on the target population, and was correlated with the state of activation of the macrophage as reflected by 5' nucleotidase activity. In spite of the protracted time course for Ia expression, all macrophage populations could be "triggered" (committed to a subsequent program of Ia expression) by exposure to the LK for as little as 2 hr. To be triggered, however, macrophages first had to go through a period of culture as adherent cells. Both the spontaneous loss and LK-dependent acquisition of Ia correlated with the functional capacity of these macrophages as antigen-presenting cells, indicating that these events are functionally significant.

Thymic maturation in vitro by a secretory product from macrophages.

Our results indicate that immature thymocytes can be induced by macrophage culture fluid (MCF) to differentiate in vitro. This maturation occurs during several days of culture but does not require DNA synthesis. It is accompanied by an augmentation of surface H-2 which precedes a loss of susceptibility to cytolysis with anti-TL and complement. Our evidence indicates that there is no physical loss of TL within the thymus. Thymocytes cultured with MCF also acquire the capacity to respond in an MLC. Differentiation was shown not to be due to interferon nor could it be reproduced by 2-ME. The stimulating molecule is distinct from the principal mitogenic protein found in MCF.

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.