Release of chondrocyte-stimulating factor by rabbit peritoneal macrophages.

Cultured rabbit peritoneal macrophages, after stimulation with lipopolysaccharides (LPS), produce a factor that induces normal rabbit articular cartilage cells (chondrocytes) to release collagenase and other neutral proteases in their culture medium. The release of the factor as well as the activation of chondrocytes can be significantly inhibited by paramethasone (10(-6) M). Rabbit peripheral blood monocytes produce this factor in smaller quantities. Activation with LPS does not enhance the release of factor any further by these cells. Lymphocytes have no direct effect on the chondrocytic protease synthesis. Furthermore, conditioned medium of activated lymphocytes failed to stimulate monocytes or macrophages in the absence of LPS. The macrophage medium exhibits mitogenic and phytohaemagglutinin-enhancing activity towards thymocytes of C3H/HeJ mice, but not against species-specific rabbit lymphocytes. The lymphocyte-activating factor, derived from a mouse macrophage cell line, P388D1 cells, or from other sources, was unable to stimulate chondrocytic protease secretion. Such specific induction of chondrocytic proteases by a macrophage-derived factor may have an important role in cartilage destruction in arthritic conditions, where synovium is only marginally involved.     

Synthesis and release of oxygenation products of arachidonic acid by mononuclear phagocytes in response to inflammatory stimuli

Evidence that macrophages secrete products that contribute to the central role played by these cells in chronic inflammation continues to accumulate. These products include hydrolytic enzymes active at either acid or neutral pH, prostaglandins, several components of the complement system, factors affecting the responses of T and B lymphocytes to mitogens, factor(s) causing increased proliferation and collagen synthesis by fibroblasts, pyrogen, and interferon. There is little information available concerning the effect of inflammatory stimuli on the secretion of these products. In the case of two types of macrophage secretory products, namely lysosomal acid hydrolases and prostaglandins, there is a marked dependence of their release on the nature of the stimuli presented to the cells. Zymosan or antigen-antibody complexes, which show potent inflammatory activity, cause the selective release of acid hydrolases from macrophages in a dose- and time-dependent fashion. These stimuli also cause the release of [³H]arachidonic acid from phospholipids, with resultant synthesis and secretion of prostaglandins. On the other hand, latex particles, which have minimal inflammatory activity in vivo, fail to cause selective release of lysosomal enzymes from macrophages and do not cause prostaglandin biosynthesis and secretion. The major products of arachidonic acid oxygenation produced by macrophages appear to be PGE₂ and 6-keto PGF₁, the stable metabolite of PGI₂.     

The macrophage as a secretory cell in chronic inflammation

Although it is clear that macrophages are always present at sites of chronic inflammation their contribution to the evolution of these lesions is not well understood. In vitro studies have shown that macrophages secrete a veriety of products on exposure to different stimuli. These include hydrolytic enzymes, active at acid or neutral pH, with known capacity for degrading tissue constituents. Lysosomal acid hydrolases are released from viable cells over a prolonged period of time by various agents known to cause, or are associated with, chronic inflammation. These agents may be nonimmunogenic substances, such as carrageenan and asbestos, which interact directly with macrophages or alternatively the products of immune reactions involving either B or T lymphocytes. These lymphocyte products include immune complexes of certain composition and the secreted products of T lymphocytes stimulated by nonspecific mitogens or specific antigens. in marked contrast biologically inactive substances such as latex particles or digestible substrates such as erythrocytes do not induce the selective release of acid hydrolases from macrophages. It is clear that although macrophages secrete abundant amounts of neutral proteinases under certain conditions this release does not occur necessarily during the release of acid hydrolases induced by inflammatory agents. The role played by acid and neutral hydrolases secreted by macrophages during the various stages of chronic inflammatory responses remains to be clarified.     

The role of macrophage activation in chronic inflammation

The macrophage is the characteristic cell type in chronic inflammatory reactions, in the rheumatoid synovium, as in other sites. When macrophages are activated, considerable synthesis of enzymes and other proteins occurs. Macrophages can be activated by (i) products of activated lymphocytes, (ii) immune complexes and (iii) the complement cleavage product C3b. Among the many consequences of macrophage activation are (i) secretion of hydrolytic enzymes, (ii) cleavage of C3 into C3a, which is cytolytic, and C3b, (iii) production of tissue thromboplastin, a powerful procoagulant, and (iv) formation of polyamine oxidase, which in the presence of appropriate substrates generates factors that lyse or limit the proliferation of tumour cells, lymphocytes and micro-organisms. The relevance of these observations to the pathogenesis of chronic inflammatory reactions is discussed.     

Differences in the induction of macrophage cytotoxicity by the specific T lymphocyte factor, specific macrophage arming factor (SMAF), and the lymphokine, macrophage activating factor (MAF)

Specific T cell factors, such as specific macrophage arming factor (SMAF), are involved in the initiation of the immune response. Induction of SMAF-producing T lymphocytes in vivo and of SMAF production by T lymphocytes in vitro is dependent on the presence of intact tumor cells, and is independent of antigen presentation by macrophages. SMAF renders peritoneal macrophages cytotoxic for tumor cells. The armed peritoneal macrophages expressed a specific cytotoxicity. However, antigen-presenting cells can trigger lymphokine-producing T lymphocytes. These T lymphocytes produce lymphokines (e.g. macrophage activating factor (MAF] that activate macrophages. The MAF-activated macrophages express a non-specific tumoricidal activity. In the present study, we investigated the difference in the induction of macrophage cytotoxicity by SMAF and MAF. The following differences were found: 1) SMAF renders peritoneal resident macrophages cytotoxic, whereas MAF could only render peritoneal exudate macrophages cytotoxic. 2) SMAF requires only a 4-h incubation with macrophages, whereas MAF activates macrophages optimally after 12 h. 3) SMAF-armed macrophages recognize only the specific target cell(s), and thus, the cytotoxicity is specific in its expression. MAF activated macrophages were non-specifically cytotoxic. 4) Lipopolysaccharide (LPS) in the culture medium did not enhance the cytotoxicity of SMAF-armed macrophages. In contrast, MAF induced tumoricidal activity was enhanced by adding LPS to the culture medium. 5) After adsorption chromatography with anti-murine interferon-gamma (IFN-gamma), the arming capacity of SMAF supernatant was not reduced, whereas the activating capacity of the MAF supernatant was significantly reduced or abrogated. After immunization of mice with allogeneic tumor cells, SMAF-producing lymphocytes were detected in the draining lymph nodes already 4 days after immunization and up to 12 days. Lymphocytes with the capacity to produce MAF were present in the draining lymph nodes 14-24 days after immunization. Our data indicate that the T cell factors SMAF and MAF can both render macrophages cytotoxic, but act in a different way and during different stages of the cellular immune response against allogeneic tumor cells.     

Activation of macrophages by products of lymphocytes from normal and syphilitic rabbits.

The production of soluble macrophage-activating factors by lymphocytes from syphilitic and normal rabbits was examined. Culture supernatants of splenic lymphocytes cultured with Treponema pallidum antigens or concanavalin A were incubated with rabbit peritoneal macrophages in vitro. The macrophage monolayers were then washed and infected with log-phase Listeria monocytogenes. Activation of the macrophages by lymphocyte products was measured by the ability of the macrophages to resist intracellular multiplication of Listeria and thus survive infection. Macrophages incubated with supernatants of unstimulated lymphocytes or T. pallidum-stimulated lymphocytes from normal rabbits were unable to resist intracellular multiplication of Listeria. Specifically stimulated lymphocytes from syphilitic rabbits and mitogen-stimulated lymphocytes from both normal and syphilitic rabbits demonstrated a clear ability to produce soluble factors which conferred upon macrophages the ability to limit the intracellular growth of the bacteria. Antigen or mitogen alone was unable to activate the macrophages; the presence of lymphocyte products was required.     

Modulation of macrophage fibronectin secretion by LPS

The secretion of fibronectin (Fn) by rat peritoneal macrophages was found to be down-regulated by LPS. A sensitive ELISA was used to quantitate both substrate-attached and supernatant Fn. Thioglycollate-elicited peritoneal exudate cells were observed to release a considerable amount of Fn during the adherence procedure for macrophage purification. After this procedure, macrophage Fn levels peaked within 2 hr and then declined steadily to baseline levels by 96 hr. Fn release by exudate cells during adherence purification was less affected by cycloheximide treatment than was subsequent Fn secretion by purified macrophages. Macrophages elicited with thioglycollate and P. acnes displayed enhanced Fn secretory activity when compared with resident unstimulated cells. Exposure to lipopolysaccharide (LPS) suppressed the levels of immunoreactive Fn in supernatants of elicited cells. This inhibition was shown to be dose-dependent and most significant after 24 hr of incubation. The inclusion of polymyxin B in the culture medium did not reverse the LPS-induced inhibition of Fn production but did prevent LPS stimulation of interleukin-1 secretion in the macrophage cultures. These observations demonstrate that Fn secretion by macrophages is regulated according to the functional state of the cell.     

Lymphocyte-induced macrophage cytotoxicity. III. Induction of specific macrophage cytotoxicity is independent of lipopolysaccharide

The induction of specific macrophage cytotoxicity by allo-sensitized T cells in vitro is shown to be independent of the presence of lipopolysaccharide (LPS). This contrasts with the induction of macrophage cytotoxicity by Macrophage Activating Factor (MAF). The specific macrophage cytotoxicity could be induced in LPS-free medium (less than l ng/ml). Addition of LPS to the macrophages did not increase the cytotoxicity. Addition of LPS-binding polymyxin B to the macrophages before and during the induction of cytotoxicity did not reduce the specific macrophage cytotoxicity. Macrophages obtained from the LPS-unresponsive mouse strain C3H/HeJ were rendered cytotoxic by the allo-sensitized lymphocytes to the same extent as the macrophages from LPS-responsive C3HeB/Fe and C57BL mice. This indicates that the induction of macrophage cytotoxicity by MAF is different from the induction of specific macrophage cytotoxicity by Specific Macrophage Arming Factor (SMAF).     

Production of macrophage-, granulocyte-, granulocyte-macrophage- and multi-colony-stimulating factor by peripheral blood cells

The specific cell sources and signals for induction of various colony-stimulating factors (CSF) in peripheral blood mononuclear cells (PBMC), purified T lymphocyte and monocyte (Mo) populations have been investigated. In the absence of exogenous activating stimuli, human PBMC, T cells and Mo failed to produce stable cytoplasmic mRNA for CSF for macrophages (M-CSF or CSF-1), for granulocytes (G-CSF), for granulocytes and macrophages (GM-CSF) and for multilineage CSF [multi-CSF, interleukin (IL) 3] and thus failed to release CSF proteins. However, after stimulation with phorbol myristate acetate and phytohemagglutinin, M-, G-, GM- and multi-CSF mRNA became detectable in PBMC, resulting in the secretion of the respective proteins. Identical culture conditions resulted in synthesis of only G- and M-CSF by purified Mo, whereas purified T lymphocytes produced GM-CSF and multi-CSF only. When Mo or T lymphocytes were exposed to recombinant human interferon-gamma or were stimulated by triggering the epitopes recognized by the monoclonal antibodies anti-Tll2 and Tll3, respectively, again disparate CSF expression patterns were found to be associated with both cell species. Moreover, IL2-receptive T lymphocytes showed the same distinct pattern of CSF secretion when activated by recombinant human IL2.     

Interleukin 1 and interferon-gamma: cytokines that provide reciprocal regulation of macrophage and T cell function

Interactions between macrophages and T cells are symbiotic, since optimal functions of both cell types require interchange of soluble mediators. Upon activation, macrophages release interleukin-1 (alias lymphocyte activating factor, leukocytic endogenous mediator, and endogenous pyrogen), a family of molecules with multivarious biological effects, ranging from induction of fever and the acute phase response to lymphocyte activation and concomitant release of interleukin-2. Interleukin-2 induces activation and replication of several subsets of precursor lymphocytes, including cytotoxic T cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells, and enhances their cytotoxic activity for tumor cells. Both interleukin-2 and leukotrienes enhance production of immune interferon (interferon-gamma) by activated T cells. Aside from antiviral activity, interferon-gamma produces a number of immunomodulatory effects, including macrophage activation (Ia induction, antimicrobial effector function, and activation of oxidative metabolism) and augmentation of NK function. Expression of Ia on accessory cell membranes is required for the initiation of many antigen-specific, T-dependent immune responses. Interferon-gamma also synergizes with a variety of microbial agents to augment macrophage tumoricidal function and enhance interleukin-1 secretion. The production of interferon-gamma appears to have a critical role in feeding back the cascade of interleukins in a loop of amplification. Both interleukin-1 and interferon-gamma modulate release of arachidonate metabolites in various cells. This cascade of cytokines, in collaboration with arachidonate oxygenation products, regulates immunity and sets the stage for many of the events underlying inflammation. Various anti-inflammatory drugs and immunopotentiators appear to act by modulating cytokine pathways.     

T cell-mediated cognate signaling of nitric oxide production by macrophages. Requirements for macrophage activation by plasma membranes isolated from T cells

Macrophage generation of reactive nitrogen intermediates (RNI) represents a major effector mechanism in anti-microbial immunity and non-septic inflammatory reactions. The induction of macrophage RNI production has been demonstrated to require at least two signals which in microbial infections can be provided by interferon (IFN)-γ and lipopolysaccharide (LPS). The current study demonstrates that, in the absence of LPS, T lymphocytes can provide cognate signal(s) which synergize with IFN-γ in stimulating macrophage RNI production, as evidenced by the ability of plasma membranes from T cell clones to activate IFN-γ-primed macrophages. Although viable resting T cells can activate IFN-γ-primed macrophages by an interaction that is antigen specific, plasma membranes from resting T cells do not activate macrophages. Plasma membranes from T cells activated by immobilized anti-CD3 were able to effectively induce RNI production in IFN-γ-primed macrophages. However, in contrast to the antigen-specific interaction of macrophages with viable resting T cells, the activation of IFN-γ-primed macrophages by membranes from activated T cells does not display antigen specificity. Plasma membranes from activated T helper T_H2 and from activated T_H1 cells were equally effective in activating IFN-γ-primed macrophages, suggesting that the dominance of T_H1 over TH2 cells in cell-mediated responses involving macrophage effectors is not a reflection of differences in their ability to interact with macrophages but rather is a reflection of their different pattern of cytokine production. These results suggest that the T cell-macrophage interaction involves reciprocal activation of both cells - an antigen-specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen-nonspecific stimulation of the macrophages.     

Soluble TNF and membrane TNF expressed on CD4+ T lymphocytes differ in their ability to activate macrophage antileishmanial defense.

In our studies of host defense against the intracellular parasite Leishmania major, we obtained evidence for a novel mechanism of macrophage activation for antimicrobial defense that involves direct cell contact between CD4+ T lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive as it does not involve secretion of lymphokines but is apparently mediated by the membrane-anchored form of tumor necrosis factor (mTNF; approximately 50-60 kd) present on the surface of the effector T lymphocytes. Furthermore, it is not cytotoxic to the host cell and its expression is antigen specific and genetically restricted. We prepared a Leishmania-specific cloned T-T cell hybridoma line 1B6 (CD4+, TH1) that expresses membrane-bound TNF but does not secrete TNF or other macrophage activators. We now report that 1B6 cells can activate antileishmanial defense in inflammatory macrophages, whereas soluble recombinant murine TNF (sTNF) alone is unable to do so. On the other hand, both 1B6 cells and sTNF can act synergistically with recombinant murine interferon-gamma (IFN-gamma, a known soluble macrophage-activating factor) in activating antimicrobial defense and NO2- release. The effects of 1B6 alone and the synergistic effects of 1B6 and IFN-gamma or sTNF and IFN-gamma are arginine dependent. These results suggest that mTNF may be more efficient than sTNF in macrophage activation and that contact with effector CD4+ lymphocytes that express mTNF may be an important mechanism of host defense.     

Comparison of MMP-2 and MMP-9 secretion from T helper 0, 1 and 2 lymphocytes alone and in coculture with macrophages

Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalling during chronic inflammatory states such as atherosclerosis formation. However, the sources and mediators of MMP upregulation need clarification. We investigated whether proinflammatory mouse T helper type 1 (Th1) lymphocytes are more active in MMP secretion than naïve Th0 or anti-inflammatory Th2 phenotypes, in the absence of specific antigenic stimulation, under baseline conditions and after contact with irradiated macrophages. We also compared the effect of Th0, Th1 or Th2 lymphocyte-conditioned medium and irradiated lymphocytes on MMP production from macrophages. Finally, we investigated whether CD40–CD40 ligand (CD40L) interactions were involved in T-cell-stimulated MMP secretion from macrophages. Under baseline conditions, MMP-2 messenger RNA (mRNA) and protein levels were greater in Th1 than Th0 or Th2 lymphocytes; MMP-9 mRNA, but not protein, was also upregulated. In the presence of irradiated macrophages MMP-2 and MMP-9 production from Th1 and Th2 was greater than from Th0 lymphocytes. Conditioned media from Th1 but not Th0 or Th2 cells increased MMP-9 secretion from macrophages. Irradiated Th1 lymphocytes stimulated both MMP-2 and MMP-9 secretion from macrophages more than irradiated Th2 or Th0 cells; this activation was independent of CD40–CD40L interaction. These findings demonstrate for the first time greater MMP secretion by Th1 than Th2 or Th0 lymphocytes and their greater ability to upregulate macrophage MMP secretion in the absence of specific antigenic stimulation. These mechanisms could promote matrix turnover in inflammatory states and, for example, promote atherosclerotic plaque rupture.     

The accumulation of inflammatory cells in synovial sheath and epitenon during adhesion formation in healing rat flexor tendons.

The accumulation of inflammatory cells in synovial tissue was studied using indirect immunofluorescence assays on cell cultures and frozen tissue sections of healing rat digital flexor tendons. Flexor tendons were collected from rats 3, 7 and 14 days after crush injury. Tendon sheath and epithenon cells were isolated by sequential enzymic digestion and cultured for 2 days. Subpopulations of synovial and inflammatory cells were identified with MoAbs against cell surface glycoproteins present on B lymphocytes (CD45), T lymphocytes (CD2, CD4, CD8), macrophages (CD14) and endothelial cells. A phagocytosis assay was also used to identify macrophages. We report a substantial increase in the number of T lymphocytes (mainly helper/inducer) and phagocytotic cells with monocyte/macrophage surface markers in tendon sheath and epitenon 3 days after crush injury. The infiltration of inflammatory cells into synovial sheath and epitenon preceded an increase in fibronectin production by tendon cells which was seen 7 days after injury. To study the interaction between T lymphocytes and synovial cells in vitro, we established synovial fibroblast-like type B cell cultures and used stimulated and non-stimulated T lymphocytes in cell binding assays. We observed increased adhesiveness between unstimulated synovial cells and synovial cells previously cultured with activated and non-activated T lymphocytes. ELISA inhibition studies have shown an increase in fibronectin production by synovial fibroblasts co-cultured with stimulated CD4+ T lymphocytes. We suggest that the presence of inflammatory cells in synovial sheath and epitenon during tendon healing induces synovial fibroblasts and epitenon cells to increase their production of fibronectin, which provides a scaffold for subsequent adhesion formation.     

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney,which aggravate puromycin aminonucleoside nephrosis

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1^rnu/rnu) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.     

The peritoneal antigen-presenting macrophage: control and immunogenic properties of distinct subpopulations

The control of the immunogenic antigen-presenting capacity of different subpopulations of thioglycollate-induced peritoneal macrophages has been investigated. The experiments revealed the existence of two major subpopulations of macrophages, only one of which was highly efficient in educating antigen-specific T cells. The other subpopulation, while highly phagocytic, was devoid of antigen-presenting capacity. Further analysis, using specific antisera directed at H-2I region gene products, revealed that the immunogenic antigen-presenting population expressed H-2I region-controlled membrane antigens. Searching for cellular elements which control the differentiation of this antigen-presenting macrophage subpopulation, it was found that its function was strictly controlled by T cells. T cell-deficient mice (nu/nu) failed to generate a functional antigen-presenting macrophage subpopulation. Transplantation of mature T lymphocytes to T cell-deprived mice restored the immunogenic function of their antigen-presenting macrophages. The results obtained suggest the existence of heterogeneity of functions among macrophage subpopulations and add a new regulatory function for T cells.     

Pathogenesis of hemophagocytic syndrome (HPS)

Hemophagocytic syndrome (HPS) is a clinicopathologic entity characterized by increased proliferation and activation of benign macrophages with hemophagocytosis throughout the reticuloendothelial system. Uncontrolled T-lymphocyte activation is responsible for increased T(H)1 cytokines secretion such as IFN-gamma, IL-12 and IL-18 that promotes macrophage activation. Genetic defects specific for cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have been identified in patients with primary HPS that are responsible for altered cell death and apoptosis induction or target killing. HPS may be secondary to malignancy, infection or autoimmune disease, and mechanisms involved are poorly understood. However, in adult-onset Still's disease, juvenile chronic arthritis and probably systemic lupus erythematosus, IL-18 might play a role in initiating macrophage activation.     

Cyclosporin A blocks the generation of alloindifferent but not of allospecific suppressive cells

The induction of alloantigen-indifferent, MHC unrestricted suppressive cells (SC) early on in human mixed lymphocyte culture (MLC) or after stimulation with suppressive T cell clones was blocked in a dose-dependent fashion by cyclosporin A (CsA). This was not prevented by the addition of the following defined lymphokines: interleukin (IL) -1, -2, -4, -5, interferon-tau or GM-CSF. The addition of MLC-conditioned medium as a source of multiple lymphokines including IL-3 also failed to reconstitute suppressive activity in the presence of CsA. In contrast, the development of allospecific, HLA-restricted SC in the same CsA-blocked MLC was not prevented or was even enhanced. These results confirm that CsA 'spares' specific SC induction late in MLC, but show that it prevents induction of non-specific suppression earlier in MLC by a mechanism presumably unrelated to blocking the secretion of interferon-tau or IL-1 to IL-5.