Lymphocyte migration patterns in autoimmune MRL-lpr/lpr mice: relationship to age, disease manifestations and lymphocyte homing receptor expression.

We have previously reported that lymphoid cells from systemic lupus erythematosus (SLE) mice with established disease migrate aberrantly. This study evaluates the abnormal lymphocyte migration patterns found in MRL-lpr/lpr (MRL/l) mice in relation to age, disease manifestations and the expression of lymphocyte homing receptors. 51chromium-labelled lymph node cells from MRL/l and from normal histocompatible CBA mice of different ages were injected i.v. into age and sex-matched CBA recipients. Diminished lymph node and increased hepatic uptake of MRL/l compared to CBA cells was evident as early as 6 weeks of age. Abnormalities in lymphocyte migration antedated the appearance of elevated antihistone antibody (AHA) levels but not the development of lymphadenopathy. Using the monoclonal antibody MEL-14, no differences in the expression of lymphocyte homing receptors between MRL/l and CBA lymph node cells were found at any age. Thus abnormalities in lymphocyte migration in MRL/l mice appear as early as six weeks and are not related to changes in homing receptor expression.

Release of galactosyltransferase from human platelets and a subset of monocytes in culture.

We show that human monocytes and platelets release considerable amounts of galactosyltransferase (GT) in serum-free culture as measured by the amount of incorporation of 3H-galactose into ovalbumin. Enzyme production was the greatest among medium-sized mononuclear cells separated by counter-current elutriation. The cells were adherent and positive for the monocyte-specific monoclonal antibody FMC-32. The activity in the monocyte fractions was not due to platelet contamination as shown from experiments in which platelets or platelet antigens were eliminated. Cell viability decreased by less than 3% during the overnight culture, and results from cell disruption experiments showed that the enzyme was not released from dead or dying cells. Cycloheximide inhibited release during 20 hours culture. Approximately 50% of the enzyme in the cell culture supernatant was pelletable at 105,000 g. Platelets released the enzyme more rapidly than did monocytes and were readily stimulated by thrombin to release more GT. Thrombin also increased monocyte GT activity after overnight incubation, but other stimulants, zymosan and lipopolysaccharide (LPS), decreased release. We conclude that GT is released into culture supernatants by platelets and by a subset of peripheral blood monocytes. These sources may account for a significant proportion of the serum enzyme and may be important in modification of extracellular carbohydrates during inflammation and coagulation.

Kinetics of macrophage recruitment and turnover in peritoneal inflammatory exudates induced by Salmonella or thioglycollate broth.

Kinetics of peritoneal macrophage turnover during infection of mice with Salmonella enteritidis or following injection with thioglycollate broth or other peritoneal stimulants has been studied. Single intravenous injections of tritiated thymidine were given and the cells were examined by autoradiography. Maximum labelling of small adherent peritoneal macrophages occurred when 3H-thymidine was given 1 d after Salmonella and the cells were harvested 1 d later. Labelled cells decreased at later times despite maintenance of high numbers of macrophages in the exudates. Results from experiments in which labelled peritoneal cells were reinjected indicated that small, monocyte-enriched, labelled cells were not the major source of the large macrophages. Similar labelling at 2 d was observed using heat-killed Corynebacterium parvum or lipopolysaccharide (LPS) as ip stimulants. Following injection of thioglycollate broth, labelled peritoneal macrophages were only detectable if 3H-thymidine was given before the stimulant. These labelled cells remained longer in the peritoneal cavity. Labelling of and numbers of blood monocytes were consistent with the promotion of monocytopoiesis by Salmonella but not by thioglycollate. The response to thioglycollate but not Salmonella was dependent on the age of the mice. Animals injected with thioglycollate 1 d before Salmonella also had decreased resistance to bacteria and low numbers of labelled peritoneal macrophages. We propose that thioglycollate may recruit from a subset of preformed monocytes and temporarily block monocytopoiesis or macrophage bactericidal activity.

Immunoregulation by macrophages. III. Prostaglandin E suppresses lymphocyte activation but not macrophage effector function during Salmonella enteritidis infection.

Peritoneal macrophages obtained during primary or secondary infection with Salmonella enteritidis differ in the proportions of subpopulations with the capacity to secrete prostaglandin E (PGE) and interleukin 1 (IL1) and have bactericidal and tumoricidal activities in vitro. Using indomethacin in vivo and PGE in vitro we have studied the regulation of subpopulations of lymphocytes and macrophages by PGE during the inflammatory reaction. Indomethacin treatment promoted clearance of the Salmonella and a 50-90% increase in macrophages recovered from the peritoneal cavity in both primary and secondary infected animals. Whilst blocking the capacity of macrophages to secrete PGE in vitro the indomethacin treatment did not alter their bactericidal (or tumoricidal) activity nor their cyclic AMP response to PGE2. A major effect of indomethacin in vivo and of PGE2 in vitro however, was on the production and expression of IL1 and IL2. Secretion of IL1 by macrophages in vitro was greatly enhanced in indomethacin treated mice and was suppressed in vitro by PGE2. Prostaglandin E2 also inhibited IL1 dependent T-lymphocyte differentiation. IL2 secretion and IL2 dependent blast cell proliferation in vitro and sensitivity of the cells to PGE2 inhibition increased through this sequence of reactions. Lymphocyte populations harvested at intervals during primary or secondary infection differed in their cyclic AMP response to PGE2 and in IL2 secretion in vitro. This may be related to changes in the proportions of lymphocyte subsets having Lyt 1+ markers. We conclude that peritoneal macrophages from Salmonella infected mice differ in their capacity to secrete PGE and IL1.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoregulation by macrophages II. Separation of mouse peritoneal macrophages having tumoricidal and bactericidal activities and those secreting PGE and interleukin I.

Macrophage subpopulations having bactericidal or tumoricidal activities and secreting interleukin I (IL1) or prostaglandin E (PGE) were identified through primary or secondary infection with Salmonella enteritidis and separated by sedimentation velocity. Bactericidal activity was measured by [3H]-thymidine release from Listeria monocytogenes and tumoricidal activity by 51Cr-release from C-4 fibrosarcoma or P815 mastocytoma cells. Macrophages with bactericidal activity were distinguished from those with tumoricidal activity a) during secondary infection when cytolytic activity occurred only at days 1-4 post injection and bactericidal activity remained high throughout and b) after sedimentation velocity separation. Cytolysis was consistently greatest among adherent cells of low sedimentation velocity, whereas cells with bactericidal activity increased in size during the infection. Tumour cytostasis (inhibition and promotion of [3H]-thymidine uptake) differed from cytolysis in that the former was more prolonged during infection and was also detected among large cells. Secretion of immunoregulatory molecules PGE and IL1 occurred maximally among different macrophage subpopulations separated by sedimentation velocity and depending on the type of stimulus used in vitro. There was an inverse correlation between IL1 production and PGE production after stimulation with C3-zymosan or lipopolysaccharide (LPS). The development of immunity during infection may therefore be dependent upon the relative proportions of effector and regulatory macrophage subpopulations and the selective effects of environmental stimuli on these functions.

Enhanced adhesion of rat neutrophils to Litomosoides carinii microfilariae in the presence of culture supernatants from mitogen-stimulated lymph node cells.

Microfilariae of the rat filarial parasite Litomosoides carinii are killed by normal rat neutrophils which adhere to them in the presence of antibody and complement. Adherence and killing were increased in the presence of the fluid from 48-hr cultures of Con A- or PHA-stimulated normal rat lymph node cells. Both normal and augmented binding were inhibited by cytochalasin B. The factor(s) responsible for increased binding was non-dialysable, susceptible to heat (56 degrees C, 30 min) and freezing and thawing. Its production was inhibited by cycloheximide. Pretreatment of neutrophils with stimulated culture supernatant for 3 hr, followed by washing, also augmented adherence. Similar pretreatment also enhanced phagocytosis of opsonized sheep erythrocytes and latex particles but did not increase candidacidal activity. These experiments suggest that cell-mediated immune reactions leading to lymphokine production may potentiate anti-filarial antibody-dependent cellular cytotoxicity and general phagocytosis by neutrophils.

I. Tumor growth in mice with depressed capacity to mount inflammatory responses: possible role of macrophages.

Studies were made of the effects of various treatments on the growth in mouse feet of isografts of two methylcholanthrene-induced fibrosarcomas: C-4, of CBA/J mice, and A-2, of A/J mice. The isografts were prepared by pronase digestion of subcutaneous tumors and were injected as unseparated cell suspensions or as tumor-cell-enriched suspensions after depletion of infiltrating host inflammatory cells. The recipient mice were untreated or treated with reserpine, sublethal whole body irradiation, cyclophosphamide, or corticosteroids. Depletion of host cells from the inoculum resulted in increased growth from the same number of tumor cells. Reserpine treatment decreased the growth of both tumors, whether unseparated or tumor-cell-enriched, and whether injected into the foot or the flank. Irradiation, cyclophosphamide pretreatment, and corticosteroid pretreatment decreased the growth of normal inocula or enriched inocula or both. The effects of cyclophosphamide and corticosteroids were apparently not due to cytotoxicity to tumor cells. Normal resident peritoneal cells increased tritiated thymidine uptake by tumor cells in vitro. Sedimentation velocity separation showed the largest cells to be the most potent. It is suggested that some hot inflammatory reaction is necessary for optimal tumor growth and that murine hosts produce not only cells with antitumor effects but also cells, possibly a subpopulation of macrophages, that potentiate tumor growth.

A mechanism of migration inhibition in delayed-type hypersensitivity reactions. II. Lymphokines promote procoagulant activity of macrophages in vitro.

Although lymphokine supernatants had no procoagulant activity per se, guinea pig peritoneal exudate cells (PEC), incubated with lymphokine supernatants or with a lymphokine fraction of 35,000 to 50,000 daltons decreased the recalcification time of platelet-poor plasma. PEC from animals immunized with BCG or with ovalbumin and incubated with the corresponding antigen had increased procoagulant activity. Similar effects were observed with PEC incubated with endotoxin. The induction of procoagulant activity by the cells was accompanied by a concomitant decrease in their ability to respond to chemotactic agents and to migrate from capillary tubes. Separation of exudate cells by sedimentation velocity indicated that large macrophages had the highest degree of procoagulant activity after incubation with lymphokines. The clotting time of Factor IX-deficient plasma was decreased by lymphokine-treated macrophages, indicating that the extrinsic clotting sequence may be activated as a result of increased tissue factor on the treated cells. Heparin abolished the ability of lymphokines to inhibit macrophage migration.

Bovine alpha-lactalbumin C and alpha S1-, beta- and kappa-caseins of Bali (Banteng) cattle, bos (Bibos) javanicus.

An electrophoretic examination is made of mild samples taken from eight Bali (banteng) cattle, Bos (bibos) javanicus, at Beatrice Hills, Northern territory, Australia. Starch-gel electrophoresis at pH 9.5 (NaOH-H3BO3 buffer) and filter-paper electrophoresis at pH 8.6 (diethylbarbiturate buffer) indicate that all samples contain a new alpha-lactalbumin variant, designated alpha-lactalbumin C. The order of mobility for bovine variants is / greater than B greater than C. The C variant differs from the common B variant in having one more amide residue (substitution or Gln for Glu). Examination of milk samples by urea-starch-gel electrophoresis at alkaline pH indicates that there is a new alpha S1-casein variant, designated alpha S1-casein EBali, present in some samples. No new kappa-casein variant is detected by this method (all samples typing as kappa-casein B). A new variant of beta-casein, designated A4, is detected by urea-starch-gel electrophoresis at low pH. The variants of milk proteins observed in this paper and in Bell et al. (1981) are discussed in relation to those of other members of the Bovinae, especially the yak, bos (Poephagus) grunniens.

Mechanism of resistance of mice to syngeneic methylcholanthrene induced fibrosarcomas.

Mice become resistant to challenge with certain fibrosarcomas when bearing a tumor graft (concomitant immunity) or after injection of a low, non-tumorigenic dose of cells (sinecomitant immunity). Resistance to footpad challenge was depressed or abolished by treatment with carrageenan, niridazole or reserpine, or by sublethal irradiation, all of which also depressed delayed-type hypersensitivity (DTH) reactions. Immune lymphocytes initiating tumor-suppressive reactions in the feet of non-immune mice were Thy-1+, Ly-1+, Ly-2- and Ly-3-. Injection of tumor cells into the peritoneal cavities of immune mice specifically elicited an influx of macrophages. There was evidence of macrophage stimulation in tumor-immune mice. In vitro, anti-tumor effector cells lacking individual tumor specificity could be detected among the resident peritoneal cells of tumor-immune mice and among peritoneal exudate cells of non-immune mice. The expression of acquired resistance to some tumors may involve reactions akin to DTH in which a specific reaction triggers an accumulation of nonspecific effectors.

Macrophage heterogeneity.

Macrophages are a mobile, functionally diverse group of cells which may be recruited and stimulated to a high degree of metabolic activity. Heterogeneity may be detected from one site to another and result from local influences, e.g. lung v. peritoneal cells, or occur within a population and arise dur to different stages of differentiation, maturation or activation or possibly from distinct cell lines. Recruitment and turnover are important determinants of the diversity of cells at any one site. In addition, anti-tumour, anti-microbial and secretory capacities of macrophages are greatly influenced by the degree and nature of stimulation possibly affecting only a subpopulation of the cells. Accessory cell activity is also a function of a minor population of macrophages which have distinct surface antigens. The sources of the heterogeneity and the interrelationship between the macrophages subpopulations remain to be determined.

Failure of immunogenic tumors to elicit cytolytic T cells in syngeneic hosts.

Immunogenic, methylcholanthrene-induced sarcomas failed to elicit the production of rapidly cytolytic T lymphocytes in syngeneic mice, although they did elicit the production of more slowly cytotoxic cells. They were, however, susceptible to attack by cytolytic T cells from allo-immune mice and, if infected with ectromelia virus, to attack by similar cells from virus-immune, H-2 compatible mice. It is suggested that the failure to elicit cytolytic T cell production may be due to the lack of an appropriate association between tumour-specific antigens and elements of the major histocompatibility complex.

Recongnition by guinea-pig peritoneal exudate cells of conformationally different states of the collagen molecule.

Guinea-pig peritoneal exudate cells were tested in vitro in the presence or absence of specific antiserum to native collagen for their capacity to discriminate between native and denatured collagens of various species. Adherent exudate cells bound denatured collagens, regardless of the origin of the collagen or the presence of serum. The binding was reduced if the cells were pretreated with trypsin. Recovery of binding was mediated by a normal serum component resembling an IgM antibody to denatured collagen. In the presence of normal serum, native collagen was only marginally bound, apparently in a non-specific manner. Uptake of native heterologous collagens was greatly increased in the presence of specific antiserum to native collagen with specificity of binding reflecting the type of collagen. Binding of denatured and native collagen occur via independent mechanisms.