Macrophage Chemotactic Activity Research Articles

Production of Macrophage Chemotactic Activity by Guinea Pig Peritoneal Macrophages

Macrophage infiltration into an inflammatory lesion is essential for phagocytosis and synthesis of numerous enzymes and effector molecules. Recent evidence suggests that the inflammatory cells themselves may regulate the inflammatory process extensively in the connective tissue through the release of biologically active mediators. However, it has not been shown whether macrophages are responsive for macrophage infiltration into an inflammatory area. In this study, it was found that activated macrophages produce a chemotactic activity for macrophages. Oil-induced guinea pig peritoneal macrophages were stimulated in vitro by LPS or MDP. The supernatants from these cultures were assayed for chemotactic activity of glycogen-induced guinea pig peritoneal macrophages in a multi-well Chemotaxis chamber containing a 5μm pore size polycarbonate membrane filter. The assay detected chemotactic activity in the supernatants from stimulated macrophage cultures. This chemotactic activity appeared within the first 12 hours after stimulation of macrophages and was heat labile and trypsin sensitive. Production of macrophage chemotactic activity was inhibited by the addition of cycloheximide to the macrophage cultures. By the release of this factor which stimulates macrophage migration, macrophages may be involved in the recruitment of macrophages themselves to an inflammatory site.

Role of angiotensin-converting enzyme in Bacille Calmette-Guérin-induced granulomatous inflammation. Increased angiotensin-converting enzyme levels in lung lavage and suppression of inflammation with captopril.

Lung lavage levels of angiotensin-converting enzyme (ACE)-like activity were increased in C57BL/6 mice with Bacille Calmette-Guérin (BCG)-induced chronic granulomatous pulmonary inflammation and splenomegaly. Contrariwise, ACE activity was not increased in lung lavage fluids of CBA mice that developed only minimal pulmonary inflammation in response to BCG. ACE-like activity correlated with the intensity of inflammation and Captopril, a specific competitive inhibitor of ACE activity, markedly suppressed the induction and maintenance of the BCG-induced inflammatory response in both lungs and spleen. It was necessary, however, to provide sustained treatment with large doses of Captopril in order to reduce the inflammatory response. After a single intraperitoneal injection of Captopril, ACE levels in lung lavage of BCG-injected mice were reduced but returned to preinjection levels or greater within 24 h. The highest dose of Captopril was more effective in reducing the lung fluid level of ACE in BCG-inflamed lungs. This suggests that sustained daily injections of Captopril were necessary to maintain reduced ACE levels. In vitro studies indicated that high concentrations of Captopril did not affect macrophage mobility or chemotactic activity for macrophages. Thus, ACE may act as a molecular mediator of BCG-induced granulomatous inflammation in the lung.

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.

Lipid-like agent from human neoplastic cells suppresses cell-mediated immunity

A lipid-like factor (LLF) of a phospholipid nature was isolated from spent cell-free media of explants and cell cultures from lipids collected from human mammary carcinoma and malignant melanoma cells. LLF modulates macrophage properties and inhibits macrophage chemotactic activity, spreading, lipopolysaccharide-induced tumoricidal activities, and macrophage migration. LLF is unique to tumor cells and is not present in detectable quantities in normal mammary epithelial cells or skin fibroblasts. LLF also inhibits human normal lymphocytes' response to mitogenic stimulation. Partial purification of LLF from human mammary carcinoma is attained by a combination of chloroform extraction and filtration through Amicon molecular membranes. LLF activity is not sensitive to trypsin, pronase, bovine spleen phosphodiesterase II, alkaline phosphatase, or ribonuclease, but it is completely inactivated with phospholipase and lipoprotein lipase.

Dissociation of macrophage accumulation and local chemotactic activity in delayed inflammatory sites

A dissociation between the in situ generation of lymphocyte-dependent macrophage chemotactic activity (MCA) and the accumulation of macrophages in peritoneal inflammatory exudates was demonstrated in rats stimulated intraperitoneally with a saline suspension of killed Listeria monocytogenes (LM). Treatment of specifically immunized animals with cobra venom factor (CoVF) erased the hemolytic activity of serum complement (C) and the generation of peritoneal MCA. Such C-deficient rats nonetheless marshaled a substantial number of monocyte-derived macrophages into LM-induced exudates. The results suggest that MCA does not have an obligatory role in the attraction of macrophages into lesions in which there is a delayed inflammatory component. CoVF not only abrogated lymphocyte-dependent MCA in antigen-induced exudates but also decreased MCA of fresh and of heated normal rat serum. The serum of venom-treated animals could not be rendered chemotactic by C activation. It remains to be determined whether lymphocyte-dependent MCA is a product of antigen-stimulated T cells or is generated extracellularly by the interaction of T-cell factors with a humoral precursor. In any event, lymphocyte-dependent MCA differs from C-dependent MCA insofar as it is inactivated by heating (30 min at 56 °C).

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.

Modifications of macrophage chemotaxis in rats bearing the transplantable tumour reticulosarcoma ‘IRE’

Macrophage chemotactic activity was studied as a marker of the anti-inflammatory effect accompanying tumour growth. An inhibiting effect of the tumour on the macrophage mobility was observed by comparing the macrophages collected from normal rats and those from animals bearing the IRE reticulosarcoma. To explain these results, it is postulated that a MIF, or a similar inhibiting factor is released by the tumour. Both the the nature and function of such a factor(s) remain to be clarified.

Lack of correlation between structural features and function of synthetic agents tested for leukocyte chemotaxis.

A number of proteins, poly-L-amino acids, oligopeptides and lipids were tested for neutrophil, eosinophil and macrophage chemotactic activity. One myoglobin preparation was active. Based on the negative findings for all other substances, primary structure, secondary structure, degree of hydrophobicity, size and charge of a molecule, could be ruled out as structural features recognized by chemotactically responding phagocytes.

Plasma-dependent chemotaxis of macrophages toward BCG cell walls and the mycobacterial glycolipid P3

BCG cell walls, associated with oil droplets in the form of emulsions in saline, generate macrophage chemotactic activity from fresh guinea pig plasma. Serum and heat-inactivated plasma were inactive, suggesting involvement of complement or fibrinogen-derived chemotactic factors. Suspensions of cell walls and oil droplets each generated chemotactic activity from plasma, and the activity of the cell wall vaccine was due to the additive effects of these two components. A mycobacterial glycolipid (P3), which is a constituent of BCG cell walls, also had plasma-dependent chemotactic activity. The results suggest that macrophage chemotaxis may be an important part of the immunopotentiating activity of these mycobacterial products.

Characterization of Macrophage Migration Inhibitory Factor Activity Produced in Vivo by a Cell-Mediated Immune Reaction in the Guinea Pig

Peritoneal fluid from the abdominal cavities of guinea pigs having delayed hypersensitivity to horseradish peroxidase (HRPO) was obtained by a lavage technique before and after i.p. challenge with antigen. Macrophage migration inhibitory factor (MIF) and macrophage chemotactic factor activities were measured in peritoneal fluids from each animal. Chemotactic activity for macrophages was maximal 24 hr after i.p. challenge and was absent thereafter. MIF activity was maximal in peritoneal fluid 24 to 48 hr after challenge. Macrophages were present in greatest numbers in peritoneal fluid 24 hr after challenge and returned almost to control levels at 48 hr. Macrophages in 48-hr fluid were larger and exhibited more intense cytoplasmic staining for nonspecific esterases when compared to those in 0-hr fluid. The m.w. of MIF obtained from culture supernatants of HRPO-stimulated guinea pig lymphocytes and 48-hr peritoneal fluid were found to be virtually identical, 58,000 and 54,000 daltons, respectively. MIF from these in vitro and in vivo sources were similarly resistant to heating at 56 degrees C for 30 min but were both destroyed by incubation with isoluble trypsin.

Desensitization: Effects on Cutaneous and Peritoneal Manifestations of Delayed Hypersensitivity in Relation to Lymphokine Production

Guinea pigs exhibiting delayed hypersensitivity may be desensitized by a single large dose of antigen administered intramuscularly. In the present experiments, desensitization with a soluble protein such as egg albumin (EA) or bovine gamma-globulin (BGG) was nonspecific in that they suppressed both cutaneous reactivity and the macrophage disappearance reaction (MDR) to unrelated antigen in doubly immunized animals. In contrast, myobacterial antigen, at the dose studied, showed limited specificity in that it could suppress the MDR but not a skin test to an unrelated antigen. Induction of an MDR was shown to be associated with the appearance of macrophage migration inhibitory factor (MIF) and chemotactic activity in the peritoneal exudate fluid. Pretreatment with desensitizing doses of mycobacteria prevented the EA-induced appearance of MIF in the peritoneal fluids. In contrast, chemotactic activity could still be recovered, even though the MDR was suppressed. Mycobacteria functioned as a specific desensitizer not only with respect to cutaneous reactivity, as stated above, but also with respect to the production of chemotatic factor by peritoneal exudate cells. In contrast, mycobacteria functioned as a nonspecific desensitizer with respect to both the MDR and MIF production. These results suggest that the MDR may be an in vivo manifestation of MIF activity, whereas the cutaneous delayed hypersensitivity reaction may be more dependent upon chemotactic activity. These observations reported here, taken in conjunction with previously described in vitro studies, suggest that desensitization is a complex phenomenon which may be the result of several different mechanisms acting separately or in concert. The apparent discrepancies in the literature as to whether or not desensitization is a specific process would appear to arise from differences in the experimental model and the particular antigen used.

Quantification of mouse macrophage chemotaxis in vitro: role of C5 for the production of chemotactic activity.

Delineation of the mechanisms of macrophage accumulation at local tissue sites will further our understanding of immunologically mediated host resistance to infectious and neoplastic diseases. Since mice are frequently used for the study of immune function, we developed a method for the quantification of mouse macrophage chemotaxis in vitro. By this method it was found that the fifth component of complement is necessary for the production of chemotactic activity in mouse serum by inflammatory agents such as endotoxin or aggregated gamma globulin. The majority of macrophage chemotactic activity produced by these agents in mouse serum can be attributed to a low-molecular-weight (ca. 15,000) chemotactic factor. The data suggest that this factor is the biologically active cleavage product of the fifth component of complement, C5a.