Generation of macrophage chemotactic activity in situ in Listeria-immune rats.

Macrophage chemotactic activity (MCA) is generated in situ in peritoneal inflammatory exudates induced by antigens of the intracellular parasite, Listeria monocytogenes. Chemotactic and chemokinetic activity is formed locally in response to an immunologically specific signal. In rats that have been immunized adoptively with thoracic duct lymphocytes (TDL) from specifically immunized donors, the production of MCA depends upon stimulation by LMA of exudate-seeking S-phase lymphocytes of their progeny. The sequential appearance, increase, and subsequent decline of MCA in the peritoneal cavity parallels the influx of lymphocytes and precedes maximal recruitment of labeled monocytes from the blood. The MCA response in peritoneal exudates induced in adoptively immunized rats correlates with the level of macrophage accumulation in the peritoneal cavity and at sites of LMA injection in the pinna of the ear. The results suggest that MCA is released locally by antigen-activated lymphocytes and imply that the factor(s) concerned has a purposeful role in the host's defense by promoting the rapid deployment and/or retention of monocyte-derived macrophages in centers of infection.     

Allogeneic restriction of the delayed inflammatory reaction in the rat.

Delayed-type hypersensitivity (DTH) to Listeria monocytogenes was measured in rats that were recipients of syngeneic, semisyngeneic, and allogeneic immune thoracic duct lymphocytes (TDL). DTH could be transferred only to recipients that shared at least one haplotype with the TDL donors. The restriction was expressed in an inability of sensitized lymphoblasts to localize efficiently at antigen injection sites in the pinna of the ear and peritoneal cavity. Failure of allogeneic lymphoblasts to extravasate in more than trace numbers into Listeria-antigen-induced exudates was reflected in an absence of other lymphocyte-mediated expressions of DTH. Thus, lymphocyte-dependent MCA was not detected in Listeria-antigen-induced peritoneal exudates borne by recipients of allogeneic immune TDL and blood monocytes were not recruited in increased numbers into such exudates as they were in exudates borne by syngeneic rats. But allogeneic restriction of the delayed inflammatory response to Listeria antigen was overcome, at least in part, when antigen-presenting macrophages of the same MHC type as the immune TDL donors were implanted in the peritoneal cavity. The results encourage the belief that the observed failure of immune TDL to transfer DTH to allogeneic recipients is related to the inability of sensitized donor T cells to recognize antigen displayed by allogeneic macrophages.     

Characterization of guinea pig macrophages: I. Mobility and maturation of peritoneal cells following inflammatory stimuli

The wide spectrum of activities of macrophages may, in part, be a reflection of their different subpopulations. Sedimentation velocity was used in separate different guinea pig macrophage populations which were then compared in assays which measure some of the parameters of inflammatory responses. Normal resident, thioglycollate-induced exudates or cells elicited by ip injection of tuberculin into BCG-sensitized animals were studied. In thioglycollate exudates the small, possibly recently derived monocytes were most responsive to chemotactic agents whereas populations with high sedimentation velocity were more phagocytic, responsive to MIF and produced more LAF. By contrast, all macrophage populations or subpopulations from sensitized animals challenged with PPD were unresponsive to lymphocyte-derived chemotactic factor, endotoxin-activated serum, or to N-formyl-l-methionyl-l-phenylalanine in chemotaxis assays. In addition, macrophages from these animals did not migrate from capillary tubes in MIF assays. The lack of migration is presumably due to the action of MIF on these cells in vivo. Cells with low sedimentation velocity (predominantly lymphocytes), however, did migrate from capillary tubes. We conclude that small cells elicited by a nonspecific stimulus are probably attracted to the inflammatory site by chemotaxins and must subsequently enlarge or mature before they are able to respond to MIF, to be activated to produce LAF, or to exhibit extensive phagocytosis.     

Biologic activity of extracts of delayed hypersensitivity skin reaction sites

Extracts obtained from skin sites of delayed hypersensitivity reactions show chemotactic activity for monocytes and lymphocytes but not neutrophils. The soluble extractable factors present at these sites have in vivo activity as well; they promote the accumulation of monocytes in peritoneal exudates and cause inflammatory reactions in the skin of nonimmunized animals. The skin inflammatory infiltrates are predominantly mononuclear and are similar to those of delayed hypersensitivity reactions in actively immunized guinea pigs. The extracts which produced these effects had no detectable MIF activity, nor permeability inducing activity in excess of that obtainable from normal skin.These monocyte and lymphocyte chemotactic factors were analyzed by sucrose density ultracentrifugation. By this technique the distribution of monocyte factors corresponded rather closely with that of the monocyte chemotactic factors obtained from an antigen-activated lymphocyte culture. Similar correspondence was obtained for the bulk of the lymphocyte chemotactic activity present in skin extracts and in culture supernatants. This suggests the possibility that the lymphokine-like substances in the skin extracts might in fact represent lymphokines. Further documentation of this point will provide a link between in vitro and in vivo manifestations of delayed hypersensitivity.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are known to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O-2) production as well as the generation of PGE2, PGF2 alpha, and TXB2 from resident, oil-elicited and thioglycollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O-2, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O-2, these cells did secrete significant levels of PGE2, PGF2 alpha, and TXB2 in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE2 and PGF2 alpha when incubated with C5a. However, production of TXB2 was not stimulated. FMLP was inactive in stimulating PGE2, PGF2 alpha, and TXB2 in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

The mediator of cellular immunity. X. Interaction of macrophages and specifically sensitized lymphocytes.

Specifically sensitized lymphocytes have a focusing influence on mononuclear phagocytes that is expressed in the local accumulation and division of macrophages in bacteria-induced exudates. This was demonstrated by injecting Listeria monocytogenes into the peritoneal cavity of normal rats immediately before the animals were transfused with thoracic duct lymphocytes from either Listeria-immune donors or donors that had been infected with the unrelated parasite, Francisella tularensis. Sensitized lymphocytes originally present in the intravenous inocula were found later in the exudates. The arrival in the inflamed peritoneal cavity of specifically sensitized lymphocytes was associated with an exuberant influx of newly formed host cells and a local proliferative response that involved both immunoblasts and macrophages. These cytokinetic observations provide a plausible explanation of the delayed inflammatory response induced by the parasite, and imply that sensitized lymphocytes contribute to the host's defence by encouraging the prompt and purposeful deployment of monocyte-derived macrophages in centers of infection. In addition to their focusing influence on mononuclear phagocytes, specifically sensitized lymphocytes or their products can enhance the metabolic and microbicidal activity of macrophages. This activation process was revealed in the ability of rats infected with L. monocytogenes or BCG to control the growth of F. tularensis at a challenge site in the testis. But resistance was expressed only when the challenge organisms were injected with killed bacteria against which the recipients had been specifically immunized. The results accord with the view that macrophages are functionally activated by an immunological mechanism, and imply that the process is triggered locally by sensitized lymphocytes which are recruited from the blood.     

The in vitro bactericidal activity of peritoneal and spleen cells from Listeria-resistant and -susceptible mouse strains

Two days after Listeria-resistant (LrR) C57BL/10 mice were infected intraperitoneally with Listeria, their peritoneal macrophages demonstrated enhanced bactericidal activity beyond that seen in susceptible (LrS) BALB/c or CBA mice. Intravenous infection had no effect on peritoneal cell activity. The induction, but not expression, of the enhanced activity was radiosensitive. There was no significant difference between the strains with respect to the number of cells or cellular composition of the exudates. No difference in the in vitro chemotactic response of cells from the two strains could be demonstrated. Therefore there seems to be recruitment to the infected peritoneal cavity of C57BL/10 mice of young, efficiently bactericidal monocytes/macrophages. On the other hand, spleen cell bactericidal activity was intrinsically superior in C57BL/10 mice compared with BALB/c mice, possibly because, as a haemopoietic organ, the C57BL/10 spleen already contains high numbers of these efficient monocytes.     

Mechanisms underlying the anti-inflammatory effects of essential fatty acid deficiency in experimental glomerulonephritis. Inhibited release of a monocyte chemoattractant by glomeruli

Injection of nephrotoxic serum into rats results in glomerular inflammation and proteinuria. Rats placed on an essential fatty acid (EFA)-deficient diet are protected from the glomerular macrophage infiltration and the ensuing proteinuria. To account for this protection, we studied EFA-deficient rats to determine if there were defects in macrophage chemotaxis. We also investigated the possibility that EFA deficiency diminishes the production of a glomerular chemoattractant for monocytes. In microchemotaxis assays EFA-deficient macrophages migrated normally. EFA-deficient serum did not appear to contain a chemotactic inhibitor. Cultured glomeruli from control and control nephritic rats were found to elaborate a chemoattractant for monocytes. This chemoattractant activity was markedly enhanced after induction of nephritis, was heat stable, was not altered by inhibition of cyclooxygenase, lipoxygenase, or platelet-activating factor, and did not depend on C or the glomerular inflammatory cell infiltrate. EFA-deficient glomeruli harvested from animals receiving injections with nephrotoxic serum produced markedly less chemoattractant activity than glomeruli from control nephritic animals. Lipid extraction of nephritic glomeruli from control animals yielded chemoattractant activity in the organic phase. Extracts of EFA-deficient nephritic glomeruli had considerably less activity. We propose that EFA deficiency attenuates glomerular inflammation by inhibiting the ability of glomeruli to produce a specific lipid monocyte chemoattractant after exposure to a nephritic stimulus.     

Effect of low-frequency ultrasound on the chemotactic and phagocytic activity of peritoneal macrophages in rats

The influence of low-frequency ultrasound on the chemotactic, ingestive and digestive activity of peritoneal macrophages in rats was studied. The intraoperative treatment of the peritoneum with ultrasound enhanced chemotactic activity 3.3-fold in comparison with that in the control animals. The digestive function of peritoneal macrophages considerably increased, the stimulation of their ingestive capacity also occurred. The activation of the phagocytic function of macrophages was observed within 7 days after a single sonar treatment. The authors believe that the stimulation of the macrophage system is probably one of the mechanisms of the sanative action of ultrasound which is used at present in purulent surgery.     

The neutrophil migration induced by tumour necrosis factor alpha in mice is unaffected by glucocorticoids

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites.     

Alteration of alveolar macrophage chemotaxis,cell adhesion,and cell adhesion molecules following ozone exposure of rats

Ozone (O₃) exposure of humans and animals induces an inflammatory response in the lung, which is associated with macrophage stimulation, release of chemotactic agents, and recruitment of polymorphonuclear leukocytes (PMNs). This study was designed to investigate the functional aspects of the macrophages that impact inflammatory processes in the lung. Macrophages recovered by bronchoalveolar lavage (BAL) from rats exposed to purified air or 0.8 ppm O₃ were studied for their chemotactic activity, adhesive interactions with alveolar epithelial cells in culture, surface morphology, and surface expression of cell adhesion molecules. The macrophages isolated from O₃-exposed rats exhibited a greater motility in response to a chemotactic stimulus than the macrophages isolated from rats exposed to purified air. The macrophages from O₃-exposed animals also displayed greater adhesion when placed in culture with epithelial cells isolated from adult rat lung (ARL-14) than the macrophages from control rats. Both chemotactic motility and cell adhesion stimulated by O₃ exposure were attenuated when the macrophages were incubated in the presence of monoclonal antibodies to leukocyte adhesion molecules, CD11b, or epithelial cell adhesion molecules, ICAM-1. Flow cytometry revealed a modest increase in the surface expression of CD11b but no change in ICAM-1 expression in macrophages from O₃-exposed rats when compared to those from the air-exposed controls. The results demonstrate an alteration of macrophage functions following O₃ exposure and suggest the dependence of these functions on the biologic characteristics, rather than the absolute expression, of the cell adhesion molecules. © 1996 Wiley-Liss, Inc.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Polyester treatment in rats with a dorsal air pouch: chemotaxis in lavage fluid from the pouch]

Minced polyester threads introduced into peritoneal cavity of rats cause a granulomatous inflammation with evidence of macrophage stimulation. Chemotactic agents play an important role in the inflammatory reaction; they are released locally by cells involved in inflammation. In this paper the chemotactic effect of the peritoneal and subcutaneous air pouch fluids from rats bearing the polyester inflammatory process, have been studied on PMN cells "in vitro". The fluids were obtained by washing the cavity of untreated rats or rats injected with polyester, 7 days after the injection. The chemotactic response was assayed by employing modified chemotaxis Boyden chambers (Blind Well Neuro Probe) and polymorphonuclear cells from normal rats. Quantification of the migration was calculated by chemotactic index (A/B) (B = random migration, A = chemotaxis). The results demonstrate that a chemotactic activity is present in peritoneal and subcutaneous air pouch fluids following the inflammatory process. In conclusion the chronic inflammation determines the appearance of chemotactic factors for PMN cells, in the peritoneal cavity and in the air pouch, and the air pouch is a very convenient experimental system with the particular advantage that it permits easy repeated sampling of exudate during the course of an inflammatory response.     

Unique determinants of alveolar macrophage spontaneous and chemokinetically stimulated migration.

The in vitro migration of rabbit alveolar and peritoneal macrophages was quantitated by an agarose well assay which permitted the distinction of chemokinetic and chemotactic patterns of stimulation by rabbit serum, tryptic fragments of the fifth component of complement, and the synthetic peptide formyl-methionyl-phenylalanyl-leucine. The peritoneal macrophages exhibited greater chemotaxis than the alveolar macrophages, but the magnitude of the chemokinetic response of both macrophage populations to each stimulus was much greater than that of the corresponding chemotactic response. Preincubation of macrophages with 2,4-dinitrophenol suppressed the spontaneous and chemokinetic migration of the alveolar macrophages without influencing the migration of the peritoneal macrophages, while iodoacetate inhibited the migration of both types of macrophages. The addition of a crude preparation of surfactant to the macrophages stimulated the migration of both the alveolar and peritoneal populations. Alveolar macrophages are thus not only uniquely adapted to the high oxygen concentrations in their environment, but may perform their surveillance of the pulmonary surfaces more efficiently as a result of the presence of surfactant or related lipoproteins.     

The role of C3 as an opsonin in the early stages of infection.

In order to investigate the role of C3 in host defense in vivo, normal AKR/J mice, genetically deficient in C5, were depleted of serum C3 by the injection of purified cobra venom factor (CoVF). Concurrent with their C3 depletion, their serum opsonizing activity decreased to a level less than 20% of normal. When these mice were challenged with an intraperitoneal injection of pneumococci 2 hr after the CoVF treatment, the LD50 was from 30 to 80 times lower than the LD50 in saline-treated control animals. When the CoVF was given only 6 hr after the pneumococcal challenge, the LD50 was the same as in the control mice. If the pneumococci were first preopsonized in vitro and then injected into CoVF-treated animals, the LD50 was the same as that in control animals. These experiments demonstrate that C3 plays a significant role in vivo in the host's defense against infection and that a major part of that role is through its action as an opsonin. Furthermore, these experiments demonstrate that the role of C3 is most significant during the early stages of bacterial invasion.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are knwon to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O₂⁻) production as well as the generation of PGE₂, PGF_2α, and TXB₂ from resident, oil-elicited and thiogylcollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O₂⁻, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O₂⁻, these cells did secrete significant levels of PGE₂, PGF_2α, and TXB₂ in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE₂ and PGF_2α when incubated with C5a. However, production of TXB₂ was not stimulated. FMLP was inactive in stimulating PGE₂, PGF_2α, and TXB₂ in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

Effects of neoplasms on inflammation: depression of macrophage accumulation after tumor implantation.

The local accumulation of macrophages at sites of neoplasms may be a critical event in immunologically mediated tumor killing. Individuals with neoplasms, however, have been noted to have depressed monocyte chemotactic responsiveness in vitro. To determine the effect of neoplasms on macrophage migration, mice were implanted subcutaneously with either sarcoma or hepatoma cells and their macrophage migratory function quantified in vivo and in vitro. The ability of tumor-bearing animals to mobilize macrophages to an inflammatory site in vivo was depressed by as much as 61% by 6 days after tumor implantation. The in vitro chemotactic responsiveness of macrophages recovered from the peritoneal cavities of tumor-bearing animals was also markedly depressed. Macrophage migration was not affected by implantation of normal syngeneic or allogeneic tissues. In addition, the accumulation of polymorphonuclear leukocytes in vivo was not depressed in tumor-bearing animals. These findings suggest that neoplasms themselves may depress the host's ability to localize macrophages at inflammatory sites in vivo and thereby hinder immunologically mediated tumor destruction.     

Porcine spermadhesin PSP-I/PSP-II stimulates macrophages to release a neutrophil chemotactic substance: modulation by mast cells

The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) alpha antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFalpha by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.     

Effect of stress-induced lipid peroxidation on functions of rat peritoneal macrophages

The aim of the present study was to investigate the effects of stress-induced lipid peroxidation on macrophages' functions. Animals were subjected to 4 h immobilization at 4 degrees C in restraining devices. The peritoneal macrophages obtained from rats exposed to cold and restraint stress exhibited an increase in lipid peroxidation and a decline of chemotaxis and phagocytosis compared with control rats. After supplementation with vitamin E, the increment in thiobarbituric acid reactive substances (TBARS) content as the oxidative stress marker and the decline of chemotaxis and phagocytosis in peritoneal macrophages observed during cold-restraint stress was significantly removed. No significant change in catalase activity of peritoneal macrophages was observed in groups exposed to cold-restraint stress and treated with vitamin E. These findings indicate that phagocytic and chemotactic capacities of peritoneal macrophages are decreased by cold-restraint stress and this effect of stress may be related to lipid peroxidation.     

Inhibition of normal rat macrophage functions by soluble tumor products

Summary The phagocytic and chemotactic activities of normal rat peritoneal macrophages were inhibited by sera from tumor-bearing rats (TBR) and 3 M KCl extracts of tumor mass. However, sera from Corynebacterium parvum- or Listeria monocytogenes-treated TBR did not inhibit phagocytosis. On the other hand, sera from C. parvum-treated, but not from L. monocytogenes-treated TBR still inhibited the chemotactic response of the normal macrophages. Furthermore, 3 M KCl extracts of tumors from C. parvum-treated TBR did not inhibit phagocytosis and chemotactic response of the same cells. Similar results were obtained with extracts of tumor masses from L. monocytogenes-treated rats. It is suggested that treatment with bacterial immunomodulators can influence the release from neoplastic cells of soluble products influencing normal macrophage functions.