Specific binding of T lymphocytes to macrophages. I. Kinetics of binding.

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.     

B1-B cells are the main antigen presenting cells in CpG-ODN-stimulated peritoneal exudate cells

Peritoneal exudate cells (PEC) have long been used as antigen presenting cells (APC), because they have been considered to contain mainly macrophages. However, it is still unclear specifically which cells of the peritoneal exudate function as APC. Herein, we focused on macrophages and B1-B cells of the PEC and examined their APC function and cytokine production. B1-B cells purified from PEC functioned effectively as APC after CpG-stimulation and mainly produced IL-10. In contrast, macrophages purified from PEC were not able to present incorporated antigens to T cells, despite the production of IL-12 and expression of co-stimulatory molecules after CpG stimulation. These results suggest that previously held ideas regarding the functions of the mixture of cells in the PEC need to re-evaluated. In summary, the antigen presenting function of PEC was mainly attributed to B1-B cells and immunoenhancing cytokine production was dominantly derived from peritoneal macrophages.     

Specific binding of T lymphocytes to macrophages. II. Role of macrophage-associated antigen.

Peritoneal exudate lymphocytes from immune guinea pigs that bind in vitro to autologous antigen-pulsed macrophages were allowed to proliferate for 1 week to give a population markedly enriched in antigen-specific T cells. This enriched population was then studied with regard to its binding to fresh autologous antigen-pulsed macrophages. Specific binding was not inhibited by a large excess of antigen in the media (5000-fold greater than the amount of antigen associated with the macrophages) either soluble or bound to Sepharose beads, or by coating the antigen-pulsed macrophags with antibody to the exogenous antigen, by reacting a second layer of antibody to the heterologous antibody, or by haptenating the antigen and treating the hapten-antigen macrophage complex with excess anti-hapten antibody. Results of treating antigen-pulsed macrophages with the proteolytic enzymes trypsin and pronase indicate that exogenous antigen is on the macrophage surface, but the experiments failed to prove that the removable antigen is essential for binding. The simplest interpretation of these results is that the T cell receptor is not specific for native exogenous antigen.     

Inhibition of growth of Histoplasma capsulatum by lymphokine-stimulated macrophages

Peritoneal cells from mice immunized by sublethal infection inhibit the intracellular growth of Histoplasma capsulatum in vitro. Lymphokines generated in cultures of immune splenocytes stimulated with Histoplasma antigen activate normal macrophages to inhibit the intracellular growth of the fungus. Such lymphokines may inhibit the intracellular growth of H. capsulatum by 60 to 80% but have no direct effect on the viability of the fungus extracellularly. The lymphokine preparations have high interferon activity that is heat stable and acid labile. The cells in the spleen that are responsible for lymphokine production are T lymphocytes, but the help of other cells such as macrophages is essential for maximal production.     

Role of macrophages in the immune response to soluble protein antigen and in staphylococcal infection]

In experiments on rabbits immunized with soluble protein antigen immune reactions were found to be accompanied by the production of lipofuscin in macrophages. This process was the morphological manifestation of the digestion of antigen by macrophages which thus acquired the ability to migrate in the organ and to form lymphoid follicules in the medullary zone of lymph nodes. The newly formed follicules seem to be the basis of pronounced specific immune response. In staphylococcal bacteriemia the phagocytic activity of macrophages was delayed, thus causing disturbances in lipofuscin production; as a result, the subsequent phases of immune response also lagged somewhat behind in time.     

Specific binding of T lymphocytes to macrophages. III. Spontaneous dissociation of T cells from antigen-pulsed macrophages.

Peritoneal exudate lymphocytes (PEL) from immune guinea pigs that adhere to antigen-pulsed macrophages (MO) were cultured for 1 week to yield a population enriched in antigen-specific (selected) T cells. These cells bind specifically within hours to fresh autologous antigen-pulsed MO. Thd dissociation of these selected PEL from antigen-pulsed MO was studied. No evidence was obtained that factors in the culture medium play a role in dissociation. Lymphocytes that have dissociated from antigen-pulsed MO are usually fully capable of rebinding to MO freshly pulsed with antigen, suggesting that there is no deficiency in the lymphocytes ability to bind. In contrast, readding antigen to cultures during incubation prevents the predicted dissociation. Moreover, repulsing MO cultured without selected PEL restores their capacity to bind fresh selected PEL. These findings indicate that decay of antigen associated with with MO is the major mechanism underlying the observed dissociation.     

IgE immune complexes induce immediate and prolonged release of leukotriene C4 (LTC4) from rat alveolar macrophages

Alveolar macrophages obtained by lung lavage from rats were incubated with monoclonal mouse anti-DNP IgE and specific antigen (DNP-HSA) and were found to release a slow reacting substance (SRS), which was characterized by high performance liquid chromatography as leukotriene C4 (LTC)4. Alveolar macrophages incubated with 1 microM A23187 (calcium ionophore) released similar amounts of SRS (6.0 +/- 2.2 and 5.7 +/- 3.7 X 10(-10) mol of LTC4 per 5 X 10(6) alveolar macrophages, respectively). The optimal conditions and mechanism of LTC release by IgE and antigen were examined. LTC4 release was maximal when freshly retrieved alveolar macrophages were incubated for 20 min with 10 micrograms/ml IgE and then for 20 min with 100 ng/ml antigen or for 20 min with IgE and antigen that had been preincubated together for 30 min at room temperature. In addition, LTC4 release was maximal when cells were challenged with IgE and antigen in a protein-free balanced salt solution and when the cells were tumbled to prevent adherence. Dose response experiments revealed that macrophages released LTC4 when stimulated with as little as 10 ng IgE and 100 ng DNP-HSA. Alveolar macrophages did not release LTC when challenged with IgE or DNP-HSA alone. Activation of LTC4 release by IgE and antigen was rapid in onset (2.5 to 5 min), and washing to remove fluid phase IgE and antigen revealed that once activated, alveolar macrophages were capable of prolonged and continuous release of LTC4. Peritoneal lavage cells stimulated with IgE and antigen did not release SRS but could release SRS when incubated with A23187 (5.7 +/- 1.3 X 10(-10) mol LTC4/5 X 10(6) macrophages). A large variability existed between individual rats in the ability of their alveolar macrophages to be activated by IgE and antigen to release LTC4. DNP-HSA labeled with 125I was used to show formation of immune complexes of IgE and antigen when IgE and antigen were incubated together before macrophage challenge. IgE immune complexes containing as little as 2 ng of antigen elicited the release of LTC4 from alveolar macrophages. These data indicate that rat alveolar macrophages release primarily LTC4 when challenged with IgE immune complexes, and that the alveolar macrophage may differ in this respect from peritoneal macrophages that do not release detectable quantities of LTC4 when challenged under identical conditions.     

Effect of C-reactive protein on peritoneal macrophages. I. Human C-reactive protein inhibits migration of guinea pig peritoneal macrophages

The effect of human C-reactive protein (CRP) isolated and purified from pooled patients' sera on macrophage function, especially on macrophage migration, was studied. Peritoneal exudate cells (PEC) from guinea pigs were used for macrophage migration inhibition (MMI) test of capillary method. Migration of either PEC or adherent purified macrophages exposed to CRP were inhibited dose-dependently. These findings indicate that CRP inhibits macrophage migration directly, not via activation of lymphocytes contained in PEC. As control, we examined the effect of normal human serum, anti C-polysaccharide antibodies isolated from patients' sera, and free endotoxin at the dose contaminated in CRP preparation on macrophage migration and found that none of them were effective. The effect of CRP on MMI of sensitized PEC exposed to antigen was also studied. Large amounts of CRP inhibited MMI induced by antigen, indicating the possibility that CRP may act on macrophages competitively with migration inhibitory factor (MIF) and may modulate MMI. CRP possesses MIF-like activity and may play a functional role at the site of tissue injury by causing the accumulation of macrophages.     

Functional changes in murine macrophages infected with cytomegalovirus relate to H-2-determined sensitivity to infection

Peritoneal macrophages were infected with murine cytomegalovirus in vitro, and indices of infection and macrophage function were monitored over 4 days. When the cells were assessed for the expression of viral antigen or for cytopathic effects, infection was found to be solely determined by the H-2 phenotype. Less than 10% of the macrophages from resistant H-2k strains were affected, whereas 90% of H-2d cells and approximately 80% of H-2b and H-2a cells became infected. Similar trends were demonstrable by the measurement of viral DNA. In H-2a cells (B10.A), Dd conferred sensitivity despite the resistant K and class II phenotype. The findings suggest a critical association between the class I antigens and an early stage in the infectious process. Indices of infection were paralleled by a loss of Fc receptor expression and optimal colloidal gold uptake, whereas most cells remained trypan blue negative, retained dehydrogenase and acid phosphatase activities, and did not release infectious virus during the period of study. This is consistent with a role for macrophages in the persistence of cytomegalovirus in the host.     

Immunological depression produced in vitro by the action of normal RNA on mice spleen cells

Macrophages and lymphocytes from normal mice spleens were treated separately with isologous RNA, pooled again and then stimulated with antigen (rat red blood cells). The number of rosette-forming cells was used as a measure of the antibody production. A low immunological response was obtained when macrophages were incubated with normal RNA. No effect was observed when macrophages were previously sensitized with the antigen and then incubated with normal RNA or when the antigen was pretreated with RNA. On the other hand, RNA had apparently no effect on lymphocytes. Macrophage phagocytosis was markedly diminished by the addition of RNA to the culture.It can be concluded that the effect of normal RNA is exerted on the macrophages by depressing their ability either to recognize or process the antigen or both. The capacity of macrophages to transfer the immunological information to lymphocytes after it has been acquired does not seem to be altered. It can be reasonably assumed, moreover, that RNA has no effect on the antigen.     

Effect of C-reactive protein on peritoneal macrophages. II. Human C-reactive protein activates peritoneal macrophages of guinea pigs to release superoxide anion in vitro

The effect of human C-reactive protein (CRP) on macrophage function was studied in an assay of superoxide anion (O2-) production. Peritoneal exudate macrophages (PEM) of guinea pigs exposed in vitro to various doses of CRP for 72 hr resulted in the development of O2- production dose-dependently, measured by increases in superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The O2--producing activity of PEM cultured without CRP, used as a control, decreased markedly in proportion to incubation time. The O2- production by PEM exposed to CRP for 18 hr when control PEM were still high in O2- production, was decreased by larger doses of CRP, while PEM cultured without CRP for 72 hr, when O2- production by control PEM was very low, followed by incubation with CRP for another 18 hr, produced O2- CRP-dose-dependently as in the case of that observed after 72-hr incubation with CRP. These results indicate that CRP is capable of activating macrophages and acts on macrophage function as a modulator. CRP possesses migration inhibitory factor (MIF)-like activity (as reported in the preceding paper) and also macrophage-activating factor (MAF)-like activity, indicating that CRP may play a functional role at the site of inflammation and tissue damage by accumulating and activating macrophages.     

B-1 cells modulate the murine macrophage response to Leishmania major infection

AIM To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major(L. major) in vitro.METHODS Peritoneal macrophages obtained from BALB/c andBALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10(IL-10) production was quantified in the cellular supernatants using an enzymelinked immunosorbent assay. The levels of the lipid mediator prostaglandin E2(PGE2) were determined using a PGE2 enzyme immunoassay kit(Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major.RESULTS We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. CONCLUSION Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.     

Lack of response of murine peritoneal macrophages to in vitro activation by muramyl dipeptide (MDP). I. Macrophage activation by MDP is species dependent

Peritoneal exudate macrophages from mice, rats, and guinea pigs were assessed using six different parameters of macrophage activation to determine whether the cells were stimulated under similar experimental conditions. Peritoneal exudate macrophages from mice, irrespective of strain, were far less responsive to a variety of in vitro stimulatory effects of N-acetylmuramyl-L-alanyl-D-isoglutamine than those from rats or guinea pigs, while no significant differences were noted with the reactivity to stimulation by endotoxic lipopolysaccharide. We conclude that macrophage activation by MDP in vitro is species dependent.     

Down-regulation of interleukin 1 production by macrophages of sarcoma-bearing mice

Peritoneal macrophages from mice bearing a transplantable methylcholanthrene-induced sarcoma produced progressively less IL 1 as tumor burden increased. The loss of activity was not explained by the production of any inhibitor of the mouse thymocyte comitogen bioassay. Immune precipitation with a polyclonal antibody confirmed the decline in IL 1 appearance. Although tumor-bearing animals lost approximately 17% of their carcass mass, the reduced production of IL 1 was not satisfactorily explained by coexistent malnutrition, since similarly depleted non-tumor-bearing mice were capable of producing IL 1. In addition to an altered IL 1 production by macrophages of tumor-bearing mice, SDS-polyacrylamide gel electrophoresis and autoradiography revealed that the pattern of secretory protein synthesis from LPS-stimulated and unstimulated peritoneal macrophages differed between tumor-bearing and control animals. Administration of LPS to tumor-bearing mice early after tumor transplantation resulted in reduced tumor growth and prevented the down-regulation of in vitro IL 1 production by peritoneal macrophages. These findings demonstrate a specific defect in IL 1 production associated with increasing tumor burden. Further studies are required to determine whether this defect in IL 1 synthesis contributes to the increased tumor growth.     

The suppressive effect of peritoneal exudate macrophages on production of antibody to sheep erythrocytes in vitro

The effects of peritoneal exudate macrophages on antibody response to sheep erythrocytes (SRBC) were investigated in mice. Peritoneal exudate macrophages obtained from mice injected intraperitoneally with proteose peptone or Corynebacterium parvum 4 days earlier had stronger ability to phagocytize and degrade SRBC than normal resident macrophages. These macrophages suppressed antibody formation to SRBC in vitro as well as in vivo. This suppression was overridden by increasing the amount of SRBC and diminished completely by pretreatment of the macrophages with iodoacetate and partly by pretreatment with 2-deoxyglucose, both known to be inhibitors of phagocytosis, but not by addition of indomethacin to the in vitro culture. These results suggest that the suppression of antibody response by peritoneal exudate macrophages was due to the increased activity of these cells as scavenger cells, resulting in a reduced amount of effective antigenic stimulation, and that it was not mediated by a prostaglandin-dependent mechanism. The scavenger function of these macrophages may be due to Ia-negative macrophages.     

The gamma-delta T-cell cytokine response to peritonitis

The inflammatory cytokine response of peritoneal and splenic γδ T-cells and macrophages to polymicrobial bacterial peritonitis was investigated. The production of intracellular Tumor Necrosis Factor- (TNF) and Interleukin-10 (IL-10) was measured using flow cytometry at 6, 24, and 48 hrs following cecal ligation and puncture or sham laparotomy. TNF- production was induced in peritoneal — but not splenic — γδ T-cells in response to both sham surgery and peritonitis. γδ T-cells demonstrated no IL-10 production. Peritoneal and splenic macrophages demonstrated earlier TNF and IL-10 responses to CLP than γδ T-cells. We conclude that γδ T-cells are local mediators of inflammation.     

Regulation of LPS stimulated ROS production in peritoneal macrophages from alloxan-induced diabetic rats: involvement of high glucose and PPARgamma

An increased occurrence of long term bacterial infections is common in diabetic patients. Bacterial cell wall components are described as the main antigenic agents from these microorganisms and high blood glucose levels are suggested to be involved in altered immune response. Hyperglycemia is reported to alter macrophages response to lipopolysaccharide (LPS) and peroxisome proliferators activated receptor gamma (PPARgamma) expression. Additionally, glucose is the main metabolic fuel for reduced nicotinamide adenine dinucleotide phosphate (NADPH) production by pentose phosphate shunt. In this work, lipopolysaccharide (LPS) stimulated reactive oxygen species (ROS) and nitrite production were evaluated in peritoneal macrophages from alloxan-induced diabetic rats. Cytosolic dehydrogenases and PPARgamma expression were also investigated. LPS was ineffective to stimulate ROS and nitrite production in peritoneal macrophages from diabetic rats, which presented increased glucose-6-phosphate dehydrogenase and malate dehydrogenase activity. In RAW 264.7 macrophages, acute high glucose treatment abolished LPS stimulated ROS production, with no effect on nitrite and dehydrogenase activities. Peritoneal macrophages from alloxan-treated rats presented reduced PPARgamma expression. Treating RAW 264.7 macrophages with a PPARgamma antagonist resulted in defective ROS production in response to LPS, however, stimulated nitrite production was unaltered. In conclusion, in the present study we have reported reduced nitric oxide and reactive oxygen species production in LPS-treated peritoneal macrophages from alloxan-induced diabetic rats. The reduced production of reactive oxygen species seems to be dependent on elevated glucose levels and reduced PPARgamma expression.     

Lactoferrin acts on I-A and I-E/C antigen+ subpopulations of mouse peritoneal macrophages in the absence of T lymphocytes and other cell types to inhibit production of granulocyte-macrophage colony stimulatory factors in vitro

The relationship between Ia antigens on mouse resident peritoneal macrophages and the ability of lactoferrin (LF) to inhibit the production of granulocyte-macrophage colony stimulatory factors (GM-CSF) from these cells was investigated. Detection of the suppressive influence of LF on release of GM-CSF from greater than or equal to 10(5) macrophages/ml/plate required that the conditioned media being assessed for GM-CSF be prepared in the presence of indomethacin and/or be preincubated with anti-ferritin antiserum to respectively stop production of E-type prostaglandins and to remove acidic isoferritin-inhibitory activities that can mask the effects of LF. Treatment of mouse macrophages with monoclonal antibodies to the I-A and I-E/C subregions of Ia antigens in a complement C-dependent cytotoxicity assay killed less than 15% of the cells, but removed all Ia antigen+ macrophages and reduced GM-CSF production by approximately 50%. LF decreased GM-CSF production by untreated macrophages by approximately 50%, but had no effect on macrophages insensitive to treatment with anti-Ia plus C. Macrophages left at 37 degrees C for 5 and 24 hr were not killed by treatment with monoclonal anti-Ia plus C and GM-CSF production by these macrophages was not suppressed by LF. Treatment of macrophages with monoclonal anti-H-2K or anti-Mac-1 plus C reduced GM-CSF production greater than 95%. Anti-I-A, -I-E/C, -H-2K, or -Mac-1, in the absence of C, had no effect on viability of macrophages or on production of GM-CSF, but anti-I-A and -I-E/C each blocked the inhibitory action of LF. Lower concentrations of these antibodies could block the action of LF when anti-I-A and anti-I-E/C were mixed together better than when they were each used separately. The removal of Thy-1.2+ cells from unseparated or adherent peritoneal cells resulted in populations of cells that were up to 100% positive for nonspecific esterase, and did not influence GM-CSF production from these cells, the reduction of GM-CSF from these cells by LF, or the reduction of GM-CSF by the removal of Ia antigen+ cells. The results were similar whether or not T cells were removed from the assay marrow by treatment with antibodies Ly-1.1, Ly-2.2, and Qa4 plus C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Zoledronic acid repolarizes tumour‐associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway

It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB‐2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour‐free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour‐associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin‐10 was drastically down‐regulated in favour of interferon‐γ production. Peritoneal macrophages and tumour‐associated macrophages of ZA‐treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour‐associated macrophages and their reverted polarization from M2 to M1 phenotype.     

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.