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D M Mosser

Publications and source records attributed to D M Mosser.

52 records · Page 3Linked to original sources
PubMed1992

Treatment of murine macrophages with interferon-gamma inhibits their ability to bind leishmania promastigotes.

The binding of leishmania promastigotes to macrophages pretreated with interferon-gamma (IFN-gamma) was compared to binding to untreated (resident) cells. IFN-gamma-treated macrophages bound fewer leishmania promastigotes than did untreated cells. The decreased binding was apparent over a wide dose range of parasite inocula when the assays were performed in the absence of exogenous complement. This decrease was specific to leishmania, since treated and untreated macrophages bound comparable amounts of immunoglobulin G- and complement-coated sheep red blood cells. Decreased parasite binding occurred early in the macrophage activation pathway. Pretreatment of macrophages with IFN-gamma for as little as 6 h, a time insufficient to induce other macrophage activation parameters, significantly reduced their ability to bind leishmania promastigotes. To determine the mechanism of this decreased phagocytosis by activated cells, macrophages were pretreated with specific inhibitors before the addition of leishmania. The binding of promastigotes to untreated (resident) macrophages was inhibited by approximately 50% by reagents that blocked either of two macrophage receptors, complement receptor type 3 (Mac-1) or a leishmania species-specific lectin-like receptor. Binding to IFN-gamma-treated macrophage populations, in contrast, was substantially inhibited only by antibody to Mac-1. Saccharides that were 50% inhibitory in the resident cell population, decreased binding by less than 10% in activated cells. The lack of saccharide inhibition by IFN-gamma-treated cells was also reflected in an inability of activated macrophages to bind to beads coated with purified leishmania lipophosphoglycan (LPG). These LPG-coated beads bound well to resident macrophages but poorly to activated cells. Thus, leishmania bind to macrophages by two distinct mechanisms, one that utilizes Mac-1 and a second mechanism that does not depend on complement and is saccharide inhibitable. These two binding mechanisms are distinct and differentially regulated in resident and activated cells.

Animals↗
PubMed1992

An assay to quantitate the binding of Rhodococcus equi to macrophages.

A Rhodococcus equi radiobinding assay has been developed using organisms labeled with 3H-uracil. These labeled organisms resemble their unlabeled counterparts with respect to colony morphology, viability, and buoyant density. Bacteria routinely incorporate between 5 x 10(-3) and 5 x 10(-2) counts per minute per colony forming unit (cfu) which in this assay allows the detection of fewer than 0.2 cfu per macrophage. Once incorporated, greater than 90% of the label remains bacterial associated for at least 4 h postlabeling. The majority of the label is trichloroacetic acid precipitable, partitions into the aqueous phase following phenol/chloroform extraction and is ethanol precipitable. RNAse treatment of the ethanol precipitate abolishes label trichloroacetic acid precipitation. This radiolabeling technique has been used to quantitate the attachment of R. equi to both murine peritoneal and equine alveolar macrophages adherent to 13 mm glass coverslips. R. equi binding is dose dependent, saturable, and specific to macrophages. Further, binding is enhanced in the presence of fresh serum. Inhibition of radiolabeled bacterial binding can be obtained by competition with cold R. equi. This radiolabeled binding assay represents a crucial step in identifying the receptors on macrophages involved in the recognition of R. equi and may help to provide information on how macrophages recognize intracellular bacteria in general.

Animals↗
PubMed1992

Leishmania promastigotes require opsonic complement to bind to the human leukocyte integrin Mac-1 (CD11b/CD18).

Previous reports have suggested that Leishmania spp. interact with macrophages by binding to Mac-1 (CD1 1b/CD18), a member of the leukocyte integrin family. To better define this interaction, we tested the ability of leishmania promastigotes to bind to purified leukocyte integrins and to cloned integrins expressed in COS cells. We show that leishmania promastigotes bind to cellular or purified Mac-1 but not lymphocyte function-associated antigen-1 in a specific, dose-dependent manner that requires the presence of serum. Binding is inhibited with specific monoclonal antibodies to Mac-1. In the absence of complement opsonization, three different species of leishmania tested fail to bind directly to any of the three leukocyte integrins. We show that binding to Mac-1 requires the third component of complement (C3). Organisms incubated in heat-inactivated serum or serum that has been immunologically depleted of C3 fail to bind to Mac-1. Because the addition of purified C3 to C3-depleted serum restores leishmania binding to Mac-1, we suggest that parasites gain entry into macrophages by fixing complement and subverting a well-characterized adhesive interaction in the immune system between Mac-1 and iC3b.

Animals↗
PubMed1992

Platelet factor 4 efficiently reverses heparin anticoagulation in the rat without adverse effects of heparin-protamine complexes.

BACKGROUND: It has been observed that the reversal of heparin anticoagulation in humans by protamine sulfate (PS) results in various adverse reactions including leukopenia, thrombocytopenia, activation of complement, increased vascular permeability, systemic hypotension, pulmonary vasoconstriction, and pulmonary edema. The purpose of this study was to compare the efficacy and effects of native platelet factor 4 (PF4) and recombinant platelet factor 4 (rPF4) with those of PS in heparin neutralization in vivo, using a rat model. METHODS AND RESULTS: Sprague-Dawley rats were anesthetized with sodium pentobarbital, and the right femoral vein and carotid artery were cannulated. For determination of activated partial thromboplastin time, platelet count, white blood cell count, and complement titer, arterial blood samples were taken before and immediately after heparin (10 units/100 g) infusion and at several time points after the infusion of the neutralizing agent (PS, 0.1 mg/100 g; PF4, 0.5 mg/100 g). In separate groups of animals, mean arterial blood pressure was monitored throughout identical protocols and the lungs were prepared for histological examination. The anticoagulant activity of heparin was effectively reversed by all of the neutralizing agents (PS, PF4, and rPF4). Platelet count (48% of initial), white blood cell count (52% of initial), complement titer (60% of initial), and mean arterial pressure (20% decrease) decreased significantly in heparinized animals receiving PS but not in those receiving PF4 or rPF4. Lung interstitium appeared normal when heparin was followed by PF4; however, interstitial edema and hemorrhage were observed with heparin-PS. CONCLUSIONS: These results suggest that PF4 efficiently reverses heparin anticoagulation in the rat without the adverse effects of heparin-protamine complexes. Therefore, rPF4 may be an appropriate substitute for PS in patients undergoing cardiovascular surgery and other procedures that require heparin anticoagulation.

Animals↗
PubMed1991

Immunosuppression induced by attenuated Salmonella. Evidence for mediation by macrophage precursors.

An attenuated aro A- strain of Salmonella typhimurium, SL3235, was previously shown to afford excellent protection in C3HeB/FeJ mice against challenge by virulent Salmonella and cross-protection against Listeria monocytogenes. In the present study, the immunologic status of immunized mice was evaluated by studying their ability to mount in vivo and in vitro plaque-forming cell responses to SRBC. SL3235-immunized mice exhibited marked suppression in their ability to generate anti-SRBC plaque-forming cell responses. Suppression was active and mediated by soluble factors as demonstrated by the capacity of immune cells to suppress the responses of normal cells in co-culture and across a membrane in transwell plates. Depletion of T cells from immune spleens did not alleviate the suppressive activity of the remaining cells. Depletion of splenic adherent cells resulted in partial alleviation of suppressive activity, demonstrating that mature macrophages are partly responsible for mediating the observed suppression. A sequential multi-step depletion procedure resulted in marked enrichment of a second suppressor cell population that was Mac1+, Thy-1.2-, surface Ig-, J11d-, non-adherent, non-phagocytic, and esterase negative. When cultured in vitro in the presence of L cell-conditioned medium, but not in the presence of Con A supernatant, these cells matured into typical macrophages within 72 h of culture. The cell population enriched for macrophage precursors (75%) retained the suppressive capacity of unfractionated splenocytes. Our data indicate that, in addition to the well-described involvement of mature macrophages in suppressing immune responses, bacterial infection may induce appearance of macrophage precursors that may also play an important regulatory role in the immune system.

Animals↗
PubMed1990

An assay to quantitate the binding of Leishmania amastigotes to macrophages.

A leishmania amastigote radiobinding assay has been developed using organisms labeled with tritiated uracil. These labeled amastigotes resemble freshly isolated unlabeled amastigotes in metabolic activity, buoyant density, morphology, viability and their ability to transform into promastigotes. Organisms routinely incorporate between 5 x 10(-3) and 3 x 10(-2) cpm per amastigote, which allows the detection of as little as 1 x 10(4) amastigotes per assay well. This radiolabeling technique has been used to quantitate the attachment of amastigotes to macrophages adherent to either 13 mm coverslips or to 96 well plates. It can also be used to screen monoclonal antibodies to macrophage surface proteins involved in amastigote binding. Once incorporated, the label remains amastigote associated, even after intact organisms have been internalized by macrophages. It remains parasite associated until the organisms have been degraded by macrophages, at which time label is released into the supernatant. Thus, a small adaptation of the binding assay can be used to compare the intracellular survival of amastigotes in macrophages following various experimental manipulations. This amastigote radiolabeling assay, therefore, represents an important step toward determining the receptors on macrophages involved in amastigote recognition and can also be used to study the degradation of intracellular pathogens by macrophages.

Animals↗
PubMed1990

Role of complement in mouse macrophage binding of Haemophilus influenzae type b.

Previous in vivo studies demonstrated that clearance of encapsulated Haemophilus influenzae from blood is associated with the deposition of C3 on these bacteria and is independent of the later complement components (C5-C9). Since clearance of encapsulated bacteria is determined by phagocytosis of bacteria by fixed tissue macrophages, we studied the interaction of H. influenzae type b with macrophages in vitro. Organisms bound to macrophages in the presence of nonimmune serum. Binding was not evident in heat-treated serum or in serum from complement depleted animals and was inhibited by F(ab')2 fragments of antibody to C3 and by blockade of the macrophage complement receptor type 3. The majority of organisms bound in the presence of complement alone remained extracellular. Antibody in the form of convalescent serum or an IgG1 monoclonal to type b capsule did not increase the total number of organisms associated with macrophages, but did increase the number of organisms ingested. Furthermore, complement enhanced antibody-mediated ingestion. This in vitro study demonstrates that complement largely mediates binding of H. influenzae to macrophages. This binding may be critical in determining the early clearance of these bacteria from blood and may be an important mechanism of defense in the nonimmune, as well as the immune host.

Animals↗
PubMed1987

Leishmania promastigotes are recognized by the macrophage receptor for advanced glycosylation endproducts.

In this paper we demonstrate the involvement of the macrophage receptor for advanced glycosylation endproducts (AGE) in the phagocytosis of Leishmania major promastigotes. Blocking of this receptor with the ligand, AGE-BSA, leads to a 50% decrease in phagocytosis relative to controls, and a comparable decrease in the respiratory burst. The inhibition of phagocytosis by AGE-BSA was specific to leishmania. The binding of zymosan or C3bi-RBC and the phagocytosis of IgG-RBC or latex beads was not affected by the presence of AGE-BSA. Blocking of both the AGE receptor and CR3 decreases leishmania binding by nearly 90%, and reduces the respiratory burst by 80%, indicating that the two receptors account for the bulk of L. tropica promastigote recognition and uptake by the macrophage.

Animals↗
PubMed1986

Leishmania species: mechanisms of complement activation by five strains of promastigotes.

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.

Agglutinins↗
PubMed1985

The mouse macrophage receptor for C3bi (CR3) is a major mechanism in the phagocytosis of Leishmania promastigotes.

We examined the role of the macrophage receptor for C3bi, the CR3, in the phagocytosis of Leishmania major promastigotes and report that M1/70, a monoclonal antibody to the CR3, inhibited the binding of leishmania to macrophages both when the assays were performed in the presence of normal serum and in its absence. In serum, leishmania activate complement and fix C3. Fixation and subsequent cleavage to C3bi occurs rapidly, and by as early as 5 min both forms of the molecule can be identified on the parasites' surface. Complement fixation results in an enhanced phagocytosis of leishmania promastigotes by mouse macrophages. In the case of L. major, 63% of this serum-enhanced binding is inhibitable by M1/70. Binding assays were also performed in the absence of serum with the use of thoroughly washed promastigotes. The addition of M1/70 inhibited binding under these conditions by 54%. Two other rat monoclonal antibodies directed against different antigens on the macrophage plasma membrane did not inhibit binding. M1/70 did not inhibit the binding of promastigotes to rat bone marrow cells, nor did it inhibit IgG-SRBC binding to mouse peritoneal macrophages. These data indicate that the inhibition observed in the presence of M1/70 was specific for the CR3 and that the macrophage receptor for C3bi plays a major role in the phagocytosis of Leishmania major promastigotes, even in the absence of serum.

Animals↗
PubMed1985

Leishmania amastigotes: resistance to complement-mediated lysis is not due to a failure to fix C3.

Amastigote forms of Leishmania major are sensitive to lysis by fresh serum, whereas those of L. donovani are resistant. To understand the basis for this resistance we have examined the interaction of complement with amastigotes of seven strains of leishmania. Complement activation was determined by measuring the ability of amastigotes to consume complement from normal serum and by identifying parasite surface-bound C3. All of the strains that were tested activated complement, including both those that are resistant and those that are susceptible to inactivation by fresh serum. Complement consumption by amastigotes was measured as a decrease in the ACH 50 titers of serum exposed to parasites. L. major, L. donovani, and L. mexicana mexicana (strain 1VLM) amastigotes decrease titers by 35.7, 33.5, and 40.3%, respectively. The binding of C3 to amastigotes was judged qualitatively by immunofluorescence and quantitatively by a C3 radiobinding assay. L. major amastigotes bind an average of 6.6 X 10(4) molecules of C3 per parasite. L. mexicana amazonensis, L. mexicana mexicana, and L. donovani bind an average of 3.9 X 10(4), 5.9 X 10(4), and 3.7 X 10(4) molecules, respectively. In all cases, C3 binding is the result of alternative pathway activation requiring Mg++ but not Ca++. Amastigotes of the disseminating strains of leishmania represent the first example of a group of protozoa that activate early complement components leading to fixation of C3, but that are resistant to inactivation by complement.

Adult↗
PubMed1984

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages.

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.

Animals↗
PubMedDate not supplied

The third component of complement (C3) is responsible for the intracellular survival of Leishmania major.

Leishmania are obligate intracellular parasites of mononuclear phagocytes. We and others have shown that the promastigote form of all species of leishmania activates complement from non-immune serum and that this activation can result in parasite lysis. This work, as well as earlier in vivo studies, suggested that complement is an important component of host defence against leishmaniasis. We now present evidence that parasite complement fixation, in addition to increasing parasite phagocytosis, is required for the intracellular survival of leishmania in macrophages. We specifically show a strong correlation between parasite C3 fixation and intracellular survival. We attribute this survival, in part, to a decrease in the magnitude of the macrophage respiratory burst which is triggered by complement-coated, as opposed to uncoated, parasites.

Animals↗
PubMedDate not supplied

Activation of murine resident peritoneal macrophages by a cell wall extract of Candida albicans.

Ethylenediamine extraction of the cell walls of Candida albicans yields an extract which possesses antigenic activity, and like other whole cell and cell wall preparations of Candida, has been shown previously to possess immunomodulatory activity. We report here results which show that non-elicited, resident peritoneal macrophages treated with the ethylenediamine cell wall extract exhibit morphological characteristics of activated macrophages. Treated cells also manifest an increased respiratory burst response and secrete elevated levels of IL-6. The treatment with the extract appears to provide a co-priming signal to the resting macrophages, since co-stimulation with interferon-gamma results in an increased level of IL-6 and IL-1. Our results also show that extract-treated cells manifest increased responsiveness to a lipopolysaccharide as measured by the production of tumour necrosis factor-alpha. These results provide an additional basis for our understanding of the immunomodulatory activity of components of the Candida cell wall.

Animals↗