An intraperitoneal injection of latex in rabbits was found to give rise to an increase in the number of macrophages at the site of inflammation and a concomitant monocytosis in the peripheral blood. The results showed that during the initial phase of the inflammatory reaction a humoral factor is present in the circulation of these animals that stimulates the monocyte production in the bone marrow in a concentration-dependent way. This factor has been called the factor increasing monocytopoiesis (FIM), in analogy with the name given to the factor previously found in mice. Rabbit FIM is cell-line specific since it has no effect on granulocyte or lymphocyte production, has an estimated molecular weight of between 10,000 and 25,000 daltons, was found to be sensitive to treatment with proteases, to be unaffected by glycosidases, and to be readily inactivated in vitro at 37 degrees C. Neither rabbit nor mouse FIM is species specific, since rabbit FIM evoked moderate monocytosis in mice and vice versa.
Cells recovered by lavage from lungs of normal guinea pigs were centrifuged on continuous density gradients of colloidal silica (Percoll). The gradient was divided into six fractions based on the banding pattern of cells. This pattern was highly reproducible from animal to animal. Cell types in the fractions were identified by morphological and cytochemical criteria and the volume of the cells was determined by measuring their diameter and tritiated water space. More than 70% of the cells put on the gradient were recovered in the six fractions and there was no selective loss of cell types. Macrophages comprised more than 95% of the cells in fractions 3, 4, and 5. These fractions were of intermediate density (1.037-1.078 gm/ml) and together contained more than 85% of the recovered macrophages. Fraction 6 (density 1.078-1.130 gm/ml) was enriched for lymphocytes and granulocytes. Macrophages in fraction 5 were smaller, had more densely staining cytoplasm, and exhibited more nonspecific cytoplasmic esterase activity than macrophages in other fractions (5 greater than 4 greater than 3 greater than 2). These results indicate that density-gradient centrifugation on Percoll is an efficient method for purifying guinea pig alveolar macrophages and demonstrate that macrophages that differ in bouyant density also differ in morphologic and cytochemical properties. In a companion paper we report that macrophages in fractions 3, 4, and 5 differ functionally as well [9].
A significant prevalence of leprosy has been demonstrated in wild Louisiana armadillos. The Texas Gulf Coast still has endemic human leprosy, and recent mores in Texas have markedly increased armadillo-human contact. Armadillos were screened by physical examination, and by ear-snip and slit-scrape technique. Animals that screened "positive" were sacrificed and necropsied under aseptic conditions. Liver, spleen, gross lesions, and four groups of lymph nodes were cultured for mycobacteria and were studied histologically. Base ratios and DNA homology with Mycobacterium leprae were determined on mycobacteria from two armadillos (and two tissues from one of these); these studies indicate that the organism found in Texas armadillos is M leprae. Twenty-one of the armadillos were leprous--4.66%. The local prevalence varied from 1.0% to 15.4%. Epidemiologic implications of these findings and the occurrence of other concomitant mycobacterial infections are discussed.
The characteristics of murine bone marrow mononuclear phagocytes in long-term cultures with embryonic fibroblast-conditioned medium were studied to determine the stage of development and state of activation of these cells. Two liquid culture systems were used: for studies on the morphology, cytochemistry, and functional characteristics at the cellular level, the cells were cultured adherent to a glass surface; and for experiments where the cells were needed in suspension (replating experiments, and studies on locomotion, intracellular killing, and cytotoxicity) use was made of Teflon culture systems. Three developmental stages of mononuclear phagocytes could be recognized easily in these cultures: monoblasts, promonocytes, and macrophages. In cultures on a glass surface, these cells grow in colonies separate from granulocytic colonies. When incubation is prolonged beyond 7-9 days, the granulocytes die, leaving pure mononuclear phagocyte cultures. Primary cultures, in which monoblasts, promonocytes, and some macrophages proliferate, can be maintained for 3-4 weeks. Calculation showed that one monoblast present on day 0 gives rise to a progeny of more than 7 X 10(3) mononuclear phagocytes by day 14; after that, the rate of proliferation declines despite the addition of fresh media. Regular replating of the cells cultured on Teflon made it possible to maintain proliferation over a period of almost 200 days. The cells in culture have the typical characteristics of mononuclear phagocytes, as judged by light microscopy, alpha-naphthyl butyrate esterase activity, lysozyme activity, presence of receptors for Fc and C3, and endocytic, microbicidal, and cytotoxic activity. The 5'nucleotidase activity, ingestion of erythrocytes via C3-receptor, locomotion, and antibody-dependent cytotoxicity indicate that the cultured bone marrow mononuclear phagocytes are more active than resident macrophages, and as active as or even more active than thioglycollate-induced macrophages. In conclusion, the population of mononuclear phagocytes in the liquid cultures of bone marrow is heterogenous with respect to developmental stage and state of activation.
Suppressor macrophages (Mφ) which can inhibit mitogen-induced lymphocyte proliferation appeared in the spleens of mice bearing transplanted MC-A fibrosarcoma cells. An analysis of the ontogeny of such Mφ revealed additional suppressor activity directed against macrophage stem cells. Treatment of spleen cell suspensions with carbonyl iron followed by centrifugation removed suppressor Mφ but did not deplete Mφ-colony forming cells (M-CFC) which could be demonstrated in soft agar culture in L-cell conditioned medium (LCM). Untreated spleen cells had normal numbers of M-CFC; phagocyte-depleted mononuclear cells showed a threefold increase in M-CFC 14 days after subcutaneous inoculation of 106 MC-A cells per mouse. Further increases in M-CFC were also evident in similar preparations on Days 21 and 28 when the M-CFC concentration reached a maximum of eight times the normal level. The Mφ which developed from the M-CFC grown in the presence of LCM were later shown to have indomethacin-sensitive suppressor activity suggesting the mediation of this phenomenon by prostaglandins. These observations suggest that locally produced phagocytic suppressor Mφ from the spleens of tumorbearing mice play important roles not only as inhibitors of lymphocyte proliferation as reported earlier, but also as regulators of monocyte-Mφ production.
The release of C3 under various conditions by cultured monocytes/macrophages isolated from burned, trauma, and septic trauma patients was determined. When monocytes were cultured for up to 14 days there was no difference in production and release of C3 by normals and patients; C3 production rose slowly at first then rose rapidly from 8-14 days of culture. C3 production continued for up to 28 days of culture. When lymphocytes were present with the monocytes for the first 24 hr of culture there were differences in C3 production by normal and patient cells at various times of culture. There were differences in C3 release by patients' 24 hour-cultured cells at certain postburn and injury days. Macrophages from burned patients released less C3 than normal cells and macrophages from septic trauma patients released more C3 than normal cells.
Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.
A qualitative and quantitative comparison of resident peritoneal macrophages (RPM) from beige (C57BL6Jbg/bg), (bg), and control black (C57BL/6J), (BL), mice has been made. Giant anomalous lysosomes as described for other leukocytes are inconsistently demonstrable. The principal morphological expression of the bg phenotype in RPM is the presence of elongated dumbbell-shaped, horseshoe-shaped, and ring-formed cytoplasmic lysosomes demonstrable by electron microscopy. Detailed analysis indicates that these are probably variations of the same basic structure, a deformed biconcave disc with differences in form resulting from their being cut in various planes of section. Additional structural complexity results from fusion between adjacent lysosomes. Thus the distinctive lysosomes of bg mouse RPM are not totally analogous to those structures described in other cell types. A morphometric analysis of control RPM compared to those from bg animals shows a significant decrease in the lysosomal volume proportion from 5.8% to 5.0% (P less than 0.02) with a corresponding increase in the mitochondrial volume proportion in cells from bg animals (P less than 0.05). There are no differences between bg and controls in regard to cellular profile areas, nuclear profile areas, nuclear volume proportions, and the volume to surface ratios of either the average cell or nucleus. This study represents the first compilation by morphometric methods of total granule content in the bg animal and demonstrates the ability of these techniques to detect significant quantitative changes in cells as structurally complex as mononuclear phagocytes.
The purpose of these studies was to determine whether various immunomodulators such as lipopolysaccharide (LPS), lymphokines with macrophage activation factor (MAF), or muramyl dipeptide (MDP) could activate the tumoricidal properties in LPS-responsive C3H/HeN and LPS-unresponsive C3H/HeJ mice. In all studies we examined the interaction of the different immunomodulators in a free form or encapsulated within liposomes (multilamellar vesicles) with alveolar macrophages (AM) and/or peritoneal exudate macrophages (PEM). In vivo infection with viable Mycobacterium bovis, strain BCG, induced the development of highly activated macrophages from C3H/HeN mice, yet only marginally activated macrophages from C3H/HeJ mice. In vitro incubation with MAF or LPS rendered AM and PEM from C3H/HeN, but not C3H/HeJ, mice tumoricidal. The failure of C3H/HeJ macrophages to respond to LPS stimulation was due to an intracellular defect. C3H/HeJ macrophages bound fluorescein-conjugated LPS to the same extent as that found for C3H/HeN macrophages. Furthermore, LPS encapsulated in liposomes activated C3H/HeN but not C3H/HeJ AM and/or PEM. Macrophages from both strains could be rendered highly tumoricidal following interaction with free MDP or following endocytosis of liposomes containing MDP or MAF. These results indicate that the inability of C3H/HeJ macrophages to respond to LPS stimulation is specific and that the activation of macrophages by different immunomodulators could occur by different pathways.
The role of bacterial endotoxin in the expression of human monocyte cytotoxicity was studied. Endotoxin contamination of all reagents and steps of the experimental procedure were assessed by the limulus amebocyte lysate (LAL) assay. Peripheral blood monocytes were separated by adherence, and cytotoxicity was measured as [3H]-thymidine release from prelabeled mKSA-TU5 murine target cells in a 48-hr assay. Human monocytes expressed appreciable levels of spontaneous cytotoxicity under LAL- conditions irrespective of the serum source employed (LAL- or LAL+ fetal bovine serum, FBS, or LAL- human cord serum, HCS). HCS gave the highest cytotoxicity levels and LAL- FBS the lowest. Polymyxin B (10 micrograms/ml), which inhibited endotoxin-induced gelation of LAL and activation of mononuclear cells for procoagulant activity, did not affect the expression of spontaneous monocyte cytotoxicity with all three sera used. Three preparations of endotoxin used in the present study (Escherichia coli, S. typhosa, S. minnesota) caused small increases in monocyte killing in micrograms per milliliter concentrations only in 5 of 19 experiments performed. Human lymphoblastoid interferon (LAL-) and phytohemagglutinin-elicited lymphokine supernatants (LAL-) enhanced the tumoricidal activity of human monocytes under LAL- conditions and were not affected by Polymyxin B. It is concluded that exposure to endotoxin is not a prerequisite for the expression of spontaneous cytotoxicity by human blood monocytes.
Studies were undertaken to determine the effect of viable organisms of Mycobacterium bovis, strain Bacillus Calmette Guerin (BCG), on cell growth characteristics and phagocytic properties of cells from surgically-induced unilaterally hydronephrotic, contralateral, and normal rabbit kidneys. A single intravenous administration of 8 X 10(8) BCG organisms was given at the time of ureteral ligation. Four days after injection, explants were removed from the hydronephrotic, contralateral, and normal kidneys. Two cell types, fibroblasts and mononuclear phagocytes, grew from these explants. BCG caused a marked increase in the rate of growth of cells from the hydronephrotic and contralateral kidneys. There was no measurable effect of BCG on cells from the normal kidney.
We examined the involvement of the T cell recognition system in hepatic granuloma formation in nude mice infected with Mycobacterium bovis BCG and subsequently injected with spleen cells. After syngeneic spleen cell transfer, granuloma was seen in nude mouse strains of several different genetic backgrounds. When allogeneic spleen cells were transferred, only those from mice that were H-2 I-region compatible with the recipients induced granuloma formation. Adoptive transfer of BCG-immunized spleen cells to splenectomized BCG-infected nude mice also showed H-2 K-end restriction. The effect of graft-versus-host (GVH) reaction on the generation of immune T cells was examined by time course observations after transfer of H-2-compatible and -incompatible allogeneic spleen cells and by cotransferring allogeneic and syngeneic spleen cells. Under these conditions, H-2-compatible sensitized T cells were generated. The fate of completely allogeneic T cells in nude mice was examined by double transfer experiments. Allogeneic spleen cells were recovered 3, but not 8 or more, days after the transfer.
C3H mice develop heavier degrees of Pneumocystis carinii pneumonia than other mouse strains tested. We have compared P. carinii pneumonia in two strains of C3H mice: C3H/HeJ mice, which are unresponsive to the effects of bacterial lipopolysaccharide (LPS), have defects in macrophage function, and have increased antibody responses to orally administered T-dependent antigens; and C3HeB/FeJ mice, which are immunologically normal. P. carinii pneumonia was induced by corticosteroids, and the intensity of the infection was judged by a semiquantitative histopathologic scoring system. Heavier degrees of infection were found in C3H/HeJ mice than in C3HeB/FeJ mice. Serum antibodies to P. carinii, measured by an indirect fluorescent antibody technique, were mainly of the IgG class in both strains of mice and varied inversely with the intensity of P. carinii infection in the lungs. Antibody levels were significantly higher in C3H/HeJ mice than in C3HeB/FeJ mice. These data suggest that C3H/HeJ have increased susceptibility to the effects of steroids of host defenses against P. carinii, and heightened serum antibody responses to the organism.
Three characteristics of the mechanisms of antibody-dependent macrophage-mediated cytolysis (ADMC) and lectin-dependent macrophage-mediated cytolysis (LDMC) in a C3H/He mouse-MM46 syngeneic tumor system were compared: the role of divalent cations, the effect of cyclic nucleotides, and the role of protease and oxygen compounds as lytic substances. Mg++, but not Ca++, was required for ADMC but neither was required for LDMC. Ca++ inhibited both LDMC and ADMC. Dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) and its agonists, such as cholera toxin, prostaglandin E1 (PGE1) and E2 (PGE2), inhibited ADMC, but had no effect on LDMC. Dibutyryl cyclic guanosine 3',5'-monophosphate (cGMP) had no effect on either ADMC or LDMC. Isoproterenol and isobutylmethylxanthine had no effect on either ADMC or LDMC. Protease inhibitors, such as pepstatin, bestatin, chymostatin, elastatinal, leupeptin, and bovine pancreatic trypsin inhibitor, did not affect either ADMC or LDMC. Tosylphenylalanyl-chloromethylketone inhibited both ADMC and LDMC, and also inhibited macrophage-tumor cell contact in both systems. Neither catalase nor superoxide dismutase clearly inhibited either ADMC or LDMC. The different sensitivities of ADMC and LDMC to pharmacological agents suggest that these two types of mediator-dependent macrophage-mediated cytolysis have partly different cytolytic processes as well as different recognition sites.
