Reactive and nonreactive lymphocytes in experimental allergic encephalomyelitis. I. Possible role of macrophage-lymphocyte interactions.

Previous studies have shown that peritoneal inflammatory exudate cells from guinea pigs with experimental allergic encephalomyelitis proliferate prominently when cultured with the sensitizing myelin basic protein. Peripheral blood lymphocytes (PBL) obtained at the same time responded little, if at all. In the present studies, recombination experiments using appropriate mixtures of peritoneal exudate macrophages and PBL show that the presence of such macrophages will not enhance in vitro reactivity of the PBL to basic protein. The oil-induced peritoneal inflammatory response appears to deplete the PBL somewhat of antigen-reactive lymphocytes, but does not totally explain the difference in in vitro responsiveness between the lymphocytes in the peritoneal exudate and in the peripheral blood.     

Chronic inflammation induces the expression of dendritic cell markers not related to functional antigen presentation on peritoneal exudate macrophages.

Phenotypic heterogeneity between macrophages in situ has been reported, using different macrophage-specific monoclonal antibodies. Microenvironmental factors, which may play an important role in inducing this heterogeneity were studied in a local inflammatory response in vivo. Our results show that peritoneal exudate macrophages, formed during Thioglycollate induced chronic inflammation, acquire expression of the dendritic cell markers NLDC-145 and 6D2. In contrast to these phenotypic characteristics, NLDC-145+/6D2+ exudate cells do not function like dendritic cells in an allogeneic-MLR, but are suppressive when compared with steady state peritoneal cells. The expression of these membrane markers on exudate cells is discussed.     

In vitro effect of Panax ginseng on phytohaemagglutinin-induced lymphocyte transformation

The ability of inflammatory peritoneal exudate cells to modify a murine lymphoproliferative response to rye grass pollen extract was investigated. Popliteal and mesenteric lymph node cells from Balb/c mice immunised with either aqueous rye or rye grass emulsified in complete Freund's adjuvant were co-cultured with 2 X 10(3)-10(5) macrophage or polymorphonuclear neutrophil (PMN)-enriched exudate cells. There was a dose-dependent increase in the response to rye grass antigen with increasing numbers of PMN. A similar enhancement was seen with up to 10(4) macrophages per culture; higher numbers were inhibitory. Lymph node cells from rye grass or ovalbumin-immune mice that had been challenged with a combination of sensitising antigen plus 10(7) PMN gave an increased antigen-specific response in vitro. Neither antigen alone, PMN nor a mixture of PMN plus heterologous antigen was active. Adoptive transfer of a similar number of peritoneal macrophages plus antigen was also without effect. These results are in accord with the suggestion that cells obtained from acute inflammatory lesions can directly influence the activity of immunocompetent lymphocytes.     

In Vitro Proliferation of T Lymphocytes from Listeria-Infected Rodents: Assay Conditions for Rat Peritoneal Exudate Cells and Characterization of an Inhibitor

Conditions favorable to [³H]thymidine incorporation into antigen-stimulated T lymphocytes from Listeria-infected rats have been established. In cultures of peritoneal exudate (T) lymphocytes purified twice with nylon-wool vigorous antigen-specific proliferation was observed within 2 days. Cultures of lymphocytes from nodes draining a subcutaneous Listeria-infection site differed in that back-ground proliferation was higher than for peritoneal exudate lymphocytes, and [³H]thymidine incorporation was maximal at day 3. A critical factor for the rate of proliferation was the lymphocyte-to-macrophage ratio; optimal cultures of peritoneal exudate lymphocytes contained 2 to 5% macrophages. Macrophages exceeding a proportion of 10% strongly, if not completely, inhibited [³H]thymidine incorporation into antigen-stimulated lymphocytes. Inhibition was associated with mononuclear cells, adherent to plastic or nylon-wool, of the stimulated or unstimulated peritoneal cavity. It was neither attributable to release of cold thymidine from macrophages nor to rapid degradation of particulate antigen by macrophages. The degree of inhibition reflected the metabolic activity of macrophages; on a cell-for-cell basis, heat-killed and glutaraldehyde-fixed macrophages were less inhibitory, and stimulated macrophages were more inhibitory than macrophages from the unstimulated peritoneal cavity.     

Surface structure of the macrophages and lymphocytes in their interaction under conditions of antigenic stimulation observed with the scanning electron microscope]

The surface structure of macrophages, lymphocytes of peritoneal exudate and lymphocytes from the lymphatic node of intact, stimulated with meat-pentone broth and induced with antigen mice was studied using raster electron microscopy. Lymphocytes outwardly did not differ from each other irrespective the source of their origin. Macrophages obtained from the peritoneal cavity of the stimulated mice morphologically differed from those isolated from intact mice. Antigenic stimulation of macrophages made it possible to investigate the dynamics of absorption and digestion of sheep corpuscles, as well as the morphology of this process. It was established that in combined cultivation of macrophages and lymphocytes under conditions of antigenic stimulation the number of contacts between lymphocytes and between lymphocytes and macrophages considerably increased. The cytoplasmic ponticulus connecting cells was formed mostly at the expense of a single finger-shaped growth of the lymphocyte membrane.     

The rapid purification of peritoneal exudate macrophages by ficoll (polysucrose) density gradient centrifugation

Macrophages can be obtained about 99 per cent pure and in a yield of about 50 per cent by ficoll (polysucrose) density gradient centrifugation of mouse peritoneal exudate cells. The macrophages appear in the lighter fractions. The lymphocytes appear in the denser fractions and can be further purified on a polystyrene bead, rayon wool column. The method also separates guinea-pig and rat peritoneal exudate cells.

Peritoneal exudate macrophages take up colloidal radioactive gold in vivo. However, macrophages differ in the ability to take up colloidal gold and the macrophages which phagocytose the most gold appear in the lightest fraction after density gradient centrifugation.

     

Role of a non-committed accessory cell in the in vivo suppression of a syngeneic tumour by immune lymphocytes.

CBA/J mice bearing a methylcholanthrene-induced sarcoma exhibited concomitant immunity to the tumour. The cellular basis for this immunity was investigated by local transfer of mixtures of lymphoid and tumour cells into the footpads of syngeneic mice. Peritoneal exudate cells obtained from tumour-bearing mice 1 day after intraperitoneal injection of saline had a marked tumour-suppressive effect, whereas normally resident peritoneal cells did not. Peritoneal exudate cells from which adherent cells had been removed ('lymphocytes') had a suppressive effect at high or low lymphocyte: tumour cell ratios in normal recipients. In irradiated (450 R) recipients the lymphocytes were suppressive at high ratios only. At low ratios the lymphocytes were suppressive only if the irradiated recipients had been partially shielded or if the lymphocytes were admixed with peritoneal exudate cells from normal mice. It is concluded that an amplifying mechanism, possibly involving macrophages, can operate in tumour cell suppression initiated by immune lymphocytes.     

Specific surface antigens expressed on activated mouse peritoneal macrophages and recognized by a novel monoclonal antibody

A hybridoma-secreting monoclonal antibody, designated 2E12D5, was prepared by fusing mouse myelomas with spleen cells from a rat immunized with BCG-elicited mouse peritoneal macrophages. Binding of the antibody to primary mouse cells and cell lines was examined by indirect immunofluorescent flow cytometry. 2E12D5 was cytotoxic and of the rat IgG2a subclass. The antibody reacted to a great extent with the BCG-induced mouse peritoneal macrophages, half the bone marrow cells, macrophage-like cell lines and thioglycollate-induced peritoneal exudate cells to some degree, but not with other cells including BCG-elicited peritoneal lymphocytes, non- or low tumoricidal peritoneal exudate cells, thymomas, myelomas and fibroblasts. Immunoblot analysis showed the antibody to bind to four major proteins, 220,000, 125,000, 105,000 and 92,000 in molecular weight from the BCG-induced peritoneal macrophage. Pretreatment of BCG-induced peritoneal macrophages with 2E12D5 and rabbit complement greatly inhibited macrophage-mediated tumour cell cytotoxicity.     

Cytophilic properties of surface immunoglobulin of thymus-derived lymphocytes

The cytophilic properties of released surface immunoglobulins of normal thymus lymphocytes and of activated thymus-derived lymphocytes (ATC) were analysed by lactoperoxidase-catalysed radioiodination in conjunction with immunological and autoradiographic techniques. Immunoglobulin from both normal T cells and ATC was cytophilic for macrophages (peritoneal exudate cells), but showed no detectable capacity to bind to either T lymphocytes or to bone-marrow-derived lymphocytes (B cells). Under the operative experimental conditions surface immunoglobulin of B cells did not show appreciable binding to macrophages. These results support the feasibility of models of collaboration between T cells and B cells which involve a soluble antigen-specific collaborative factor (T-cell Ig complexed with antigen) and show an obligatory requirement for macrophages.     

Suppression of anti-hapten (TNP) antibody response by suppressor T cells and their product. The role of macrophages.

The effect of the lymphocytes from picryl sulfonic acid treatment animals on primary and secondary humoral immune response to TNP hapten was investigated. These cells known to inhibit contract sensitivity reactions were also able to depress primary IgM response. When peritoneal exudate cells from normal mice incubated in suppressor supernatant were transferred into primed mice the suppression of IgM but no IgM secondary response was observed. In contrast peritoneal exudate cells incubated in control supernatant augmented IgM but didn't affect IgG response. It is postulated that normal peritoneal exudate cells (presumably macrophages) can stimulate IgM response. This enhancing activity could be partially blocked by soluble suppressor factor.     

The role of resident and exudate macrophages in multinucleate giant cell formation.

Peroxidase cytochemistry which differentiates "resident" from "exudate" peritoneal macrophages in guinea pigs, was used in the investigation of the multinucleate giant cells in foreign body granulomas in the peritoneal cavity of guinea pigs. Only a few, small syncytia (two to three nuclei) displayed the cytochemical characteristics of "resident" macrophages. On the other hand, most of the polykarya at the site of inflammation displayed a distribution of peroxidase activity similar to that of "exudate" macrophages. Irrespective of whether peroxidase cytochemistry distinguishes between a distinct type of macrophage or a specific functional stage, the evidence indicates that fusion is much more frequent between macrophages with "exudate" characteristics. Thus, fusion of these latter cells is responsible for the vast majority of multinucleate giant cells in inflammatory sites. In addition, some form of recognition mechanism between macrophages of similar cytochemical characteristics probably also operates since syncytia exhibited characteristics of either "exudate" or "resident" macrophages, but never both.     

Elimination of resident macrophages from the livers and spleens of immune mice impairs acquired resistance against a secondary Listeria monocytogenes infection.

During a secondary Listeria monocytogenes infection in mice, the bacteria are eliminated more rapidly from the liver and spleen than during a primary infection. This acquired resistance against a secondary infection is dependent on T lymphocytes, which induce enhanced elimination of bacteria via stimulation of effector cells such as neutrophils, resident macrophages, exudate macrophages, and hepatocytes. The aim of the present study was to determine the role of the resident macrophages in acquired resistance against a secondary L. monocytogenes infection in mice. Mice which had recovered from a sublethal primary infection with 0.1 50% lethal dose (LD50) of L. monocytogenes intravenously (i.v.), i.e., immune mice, received a challenge of 1 LD50 of L. monocytogenes i.v. to induce a secondary infection. At 2 days prior to challenge, immune mice were given an i.v. injection of liposomes containing dichloromethylene-diphosphonate (L-Cl2MDP) to selectively eliminate resident macrophages from the liver and spleen. Control immune mice received either phosphate-buffered saline (PBS) or liposomes containing PBS (L-PBS). Treatment of mice with L-Cl2MDP effectively eliminated resident macrophages from the liver and spleen but did not affect the number of granulocytes, monocytes, or lymphocytes in peripheral blood or their migration to a site of inflammation. Phagocytosis and killing of L. monocytogenes by peritoneal exudate cells elicited with heat-killed L. monocytogenes were similar in all groups of immune mice. On day 3 of a secondary infection, the number of L. monocytogenes organisms in the livers and spleens of L-Cl2MDP-treated immune mice was 4 log10 units higher than in immune mice treated with PBS or L-PBS. The concentration of reactive nitrogen intermediates in plasma, a measure of the severity of infection, was 70-fold higher for L-Cl2MDP-treated immune mice than for PBS- or L-PBS-treated immune mice. Treatment with L-Cl2MDP significantly increased the number of inflammatory foci in the liver and spleen, decreased their size, and affected their structure. From these results, we conclude that resident macrophages are required for the expression of acquired resistance against a secondary L. monocytogenes infection in mice.     

Different effects of phytohemagglutinin-activated lymphocytes and their culture supernatants on macrophage function.

When rabbit peritoneal exudate cells were incubated for 24 and 48 h with phytohemagglutinin-activated lymphocytes or their culture supernatants, two times as many cells remained adherent to culture slides as in the controls. More spreading cells were found among the adherent cells in the stimulated cultures. Eighty percent of spreading cells that were induced by supernatants were negative or faintly positive for beta-galactosidase. On the other hand, half of the spreading cells induced by activated lymphocytes were positive (1+ to 4+) for beta-lymphocytes and their supernatants. Under similar conditions, unstimulated peritoneal cells showed less marked activation. These findings show that macrophages can appear morphologically activated and yet not be enzymatically activated by lymphokines. Possible mechanisms of direct interaction of activated lymphocytes and macrophages are discussed.     

Immune interferon. I. Production by lymphokine-activated murine macrophages.

Unfractionated murine spleen cells produce immune interferon (type II) upon stimulation with antigen or mitogen. When spleen cells were passed over glass bead columns, interferon production decreased whereas the mitotic response to the stimulants drastically increased. When these cells were further purified over nylon wool columns, interferon production was totally abolished whereas thymidine incorporation in stimulated cultures was invariably high. Interferon production by nylon wool column-purified lymphocytes could be restored with macrophages grown from bone marrow cultures or spleen cells but not with macrophages from proteose peptone-induced peritoneal exudate cells. It was also found that pure macrophage cultures from spleens of BCG-immunized mice consistently produced interferon activity without any further stimulation. When culture supernatants of activated T lymphocytes, which did not contain any interferon activity, were transferred to macrophage cultures from different sources and incubated for 45 h, interferon activity could be detected in supernatants of macrophage cultures from bone marrow and spleen but not in those from proteose-induced peritoneal exudate cells. It is concluded that certain macrophage populations can be induced to produce interferon activity whereas others are refractory to this induction which appears to be linked to their differentiation state.     

家兔实验性腹膜炎期间腹水细胞的组织化学观察

本文以腹腔注射松节油致腹膜炎后1、3、8、14 d,分别以瑞氏染色和组织化学方法对急性非特异性化学炎症期间(实验组,雄性家兔50只)渗出的腹水细胞和正常家兔(对照组,雄性家兔10只)腹水细胞进行了动态观察。瑞氏染色下,正常家兔腹水细胞为小淋巴细胞(87.1%),少量巨噬细胞(9.7%)和极少数多形核粒细胞。炎症后1 d,多形核粒细胞数目明显增高,3 d后逐渐下降。炎症后巨噬细胞数日逐渐增高,14 d后达到高峰,至30 d后仍未降至正常水平。淋巴细胞于炎症8 d后开始相对增多,14 d后尚未回至正常。组织化学观察:多形核粒细胞的碱性磷酸酶(AlP)活性,于炎症后第l d呈强阳性,3 d开始减弱。巨噬细胞的酸性磷酸酶(AcP)活性,炎症后3 d增强,5 d最强,8 d后开始减弱。脂类反应可见多形核粒细胞与巨噬细胞均呈阳性,巨噬细胞3 d反应增强,同时在胞质内存有未着色的空泡。T淋巴细胞的酸性醋酸萘酯酶(ANAE)反应,5 d时阳性反应率明显增高(从2%升至19.5%)。观察表明,在松节油所致家兔急性非特异性腹膜炎期间各种腹水细胞比例改变,功能增强,提示在炎症期间有促进免疫功能的作用。     

Biochemical and pathological features of cadmium induced peritonitis in rats.

The intraperitoneal injection of cadmium chloride (1 mg/kg of the ion) twice a day at 12-hr intervals for a maximum period of 3 days produced a severe inflammatory reaction. This reaction was characterized by an increasing accumulation of the exudate, erythrocytes and inflammatory cells into the peritoneal cavity. The peak leucocyte infiltration appeared to occur in two phases. The first occurred at 48 hr and the second at 96 hr following the initial injection of cadmium. Neutrophils always constituted 48 to 56% of the total leucocytes. Infiltration of other cell types included macrophages, monocytes, lymphocytes, mast cells, eosinophils and basket cells. The cell free supernatant of the peritoneal exudate contained the highest amount of histamine at 24 hr and the lowest amount at 48 hr after the initial injection of cadmium. The supernatant also contained an increasing amount of total PGE₂ and PGF_2α and total β-glucuronidase and lactic dehydrogenase activity in relation to the time following the initial injection of cadmium. The cyclic nucleotide level in the cell free supernatant of the peritoneal exudate was not significantly altered at the early phase of inflammation. This was however, drastically increased both at 72 and 96 hr following the initial injection. In fact, a 50-fold increase in c-AMP level and 8-fold increase in c-GMP level were noted at 96 hr as compared to their respective levels at 24 hr. The data collected in the present study suggest that the release of histamine, lysosomal enzymes, prostaglandins and cyclic nucleotides are somehow involved in cadmium induced peritonitis in rats.     

T cells expressing both L-selectin and CD44 molecules increase in number in peritoneal exudate cells and in vitro-stimulated spleen cells from mice immunized intraperitoneally with Listeria monocytogenes.

L-selectin, which was first reported as MEL-14 antigen in mice, is a type of animal lectin and expressed on lymphocytes, neutrophils and macrophages. L-selectin has been reported to be a homing receptor of lymphocytes to peripheral lymph nodes and to have an important role in initial adhesion of lymphocytes and neutrophils to endothelial cells activated by inflammatory cytokines. On the other hand, it has been reported that naive T cells express L-selectin while memory T cells and in vitro antigen-stimulated T cells lose its expression. If all memory T cells lack L-selectin, trafficking of memory T cells into inflammatory sites would be difficult. To know whether all memory T cells lack L-selectin expression, kinetics of expression of L-selectin was analysed on memory T-cell subsets, which are detected by expression of CD44, in mice after intraperitoneal immunization with a sublethal dose of viable Listeria monocytogenes. T cells expressing both L-selectin and CD44 were detected in splenocytes and peritoneal exudate cells (PEC) from untreated mice, though at low levels. L-selectin+ CD44+ T cells increased in PEC, which are known to be highly enriched in antigen-primed T cells, and reached maximum level on day 14 after immunization. Furthermore, we found increases not only of L-selectin- CD44+ but also of L-selectin+ CD44+ T cells by in vitro Listeria antigen stimulation of Listeria-immune spleen cells on day 14. These results showed that T cells expressing both L-selectin and CD44 increase after antigen stimulation in vivo and in vitro. The L-selectin+ CD44+ T cells may be a subset of memory T cells which retain their capacity of trafficking to inflammatory sites.     

The carrier effect in the secondary response to hapten-protein conjugates. 3. The anatomical distribution of helper cells and antibody-forming-cell-precursors

The activity of cells from blood, peritoneal exudate, thoracic duct lymph, lymph nodes and spleen were tested in the adoptive secondary response of mice to hapten-protein conjugates. No correlation of helper or antibody-forming-cell-precursor activity was found with the number of macrophages. Thoracic duct lymphocytes tended to be relatively more active than spleen cells as helpers. These findings are interpreted as support for the hypothesis that helper cells are thymus-derived, recirculating lymphocytes.     

Shedding light on vascular permeability during peritonitis: role of mast cell histamine versus macrophage cysteinyl leukotrienes

The inflammatory response consists of sequential steps that are essentially the same whatever the cause and wherever the site. The main purpose of inflammation is to bring fluid, proteins, and cells from the blood into the damaged tissues. Therefore there are mechanisms that allow cells and proteins to gain access to extravascular sites, where and when they are needed if damage and infection has occurred. A critical process for formation of inflammatory exudate is an increase in permeability of local blood vessels. Vasopermeability changes can be usually attributed to mast cells and their mediators but recent studies reveal that also macrophages can be involved in this process. This short commentary discusses new data on cellular origin of major vasoactive mediators, and their receptors during peritoneal inflammation in mice.     

Resident peritoneal leukocytes are important sources of MMP-9 during zymosan peritonitis: superior contribution of macrophages over mast cells

Metalloproteinase 9 (MMP-9) is crucial for normal neutrophil infiltration into zymosan-inflamed peritoneum. During the course of zymosan peritonitis MMP-9 is produced in a biphasic-manner as its presence is detectable as early as 30 min post zymosan and then between 2 and 8 h of inflammation. As inflammatory leukocytes were shown to produce MMP-9 we asked if also resident leukocytes, mast cells and macrophages, contribute to its production. And furthermore, if their contribution is limited only to the early phase of inflammation or extends to the later stages. For this purpose some mice were depleted of either resident macrophages or functional mast cells and expression of MMP-9 in peritoneal leukocytes and its release to the exudate were monitored. It turned out that depletion of peritoneal macrophages decreased both MMP-9 content in the leukocytes and its release to the inflammatory exudate at 30 min and 6h of peritonitis. The functional depletion of mast cells also caused a significant decrease in the production/release of MMP-9 that was especially apparent at the early time point (30 min). Moreover, the study shows concomitant kinetics of MMP-9 expression in leukocytes and its release to the exudatory fluid. The findings indicate that resident tissue leukocytes, and among them especially macrophages, constitute an important source of MMP-9 during acute peritoneal inflammation. Overall, the study shows that resident tissue leukocytes, mostly macrophages, constitute an important cellular source(s) of inflammation-related factors and should be regarded as possible targets of anti-inflammatory treatment.