Phagocytosis, killing, and oxidant production by bovine monocyte-derived macrophages upon exposure to Brucella abortus strain 2308

Phagocytosis and intracellular survival of Brucella abortus, and oxidant production by monocyte-derived macrophages from ten B. abortus-naive cows were studied. Phagocytosis of bacteria opsonized with naive-autologous sera or reactor serum was significantly less than phagocytosis of bacteria opsonized with fetal bovine serum. After phagocytosis, intracellular survival of bacteria opsonized with naive-autologous or reactor sera was significantly less than survival of bacteria opsonized with fetal bovine serum. Production of oxidant by macrophages stimulated with B. abortus opsonized with naive-autologous, reactor, or fetal bovine sera was not significantly different. Although macrophages from one animal showed significantly less phagocytic activity, intracellular killing and oxidant production by macrophages from the ten individual cows toward B. abortus opsonized with naive-autologous, reactor, and fetal calf sera were homogeneous. The abilities of the macrophages to phagocytize and to kill B. abortus were not associated with each other or with oxidant production. Innate resistance or sensitivity to B. abortus was not identified in the cows based on macrophage function.     

Specific anti-hairy cell and anti-B cell antisera: characterization of surface antigens and origin of hairy cells

Hairy cells obtained from nine patients with hairy cell leukaemia were found to be sensitive to a heterologous anti-human B lymphocyte serum using a cytotoxicity assay and the ultrastructural characterization after immunoperoxydase labelling. This antiserum raised in the rabbit and rendered specific by extensive absorptions with human immunoglobulins, erythrocytes, thymocytes and monocytes, reacted with normal and pathological B lymphocytes but not with monocytes, as demonstrated by ultrastructural studies. In addition, a heterologous anti-hairy cell serum was prepared and absorbed with erythrocytes, thymocytes and monocytes. The in vitro properties of this antiserum were identical to those of the anti-human B cell serum in the various assays: cytotoxicity, rosette inhibition and ultrastructural characterization. These results demonstrate that hairy cells of the studied patients express surface antigenic specificities of the B cell population, not shared by monocytes. Further absorption of the anti-hairy cell serum with CLL cells suggested that hairy cells express other characteristic antigens in addition to the B lymphocyte antigens. HLA-DR alloantigens were also shown to be present at the surface of hairy cells. This type of immunological analysis may prove to be of help in the understanding of the differentiation abnormalities in the hairy cell leukaemia as well as in other lymphoproliferative disorders.     

Nutrition education in the medical school: factors critical to the development of a successful program

The phagocytosis by mononuclear phagocytes, neutrophils and eosinophils of mast cell granules which are released in the course of anaphylactic reactions was studied in the rat. Degranulation of rat peritoneal mast cells was induced either in vivo or in vitro after passive sensitization with homologous reaginic antiovalbumin serum by challenge with the antigen. The approximate extent of degranulation was assessed by determining histamine release. The anaphylactic reaction was stopped by fixation with glutaraldehyde and the cells were examined by electron microscopy. Phagocytosis was quantified in randomly selected thin section at the magnification of 1,800. Rapid and extensive phagocytosis of mast cell granules was observed both in vivo and in vitro. About one third of the mononuclear phagocytes and between 30 and 53% of the neutrophils present were engaged in phagocytosis and usually contained several mast cell granules. Phagocytosis by eosinophils was less prominent, both with respect to the proportion of phagocytosing cell (10-23%) and to the number of mast cell granules per cell profile. Examination of large numbers of cells indicates that the uptake process is highly efficient since both condensed and already disaggregated granule bodies were seen to adhere to the phagocytes and were taken up rapidly and without the need for opsonization. In the neutrophils, extensive fusion of azurophil granules (as evidenced by peroxidase cytochemistry) with phagosomes containing mast cell granules was observed. Occasionally, mast cell granules were seen within disrupted vacuoles, which could result from the swelling of the granule matrix following engulfment. The result of this study indicate that mononuclear and polymorphonuclear phagocytes have the capacity to scavenge important amounts of mast cell granule products released by anaphylaxis.     

Cross-reactive and group-specific immune responses to a neutralizing epitope of the human respiratory syncytial virus fusion protein

Immune responses to a synthetic peptide corresponding to amino-acids 205-225 of the fusion protein from group B respiratory syncytial (RS) virus, were studied in mice and rabbits, and compared to a similar peptide from group A RS virus. Peptide 205-225 (B) was recognized by monoclonal antibody RS-348, and was immunogenic in both mice and rabbits, as was peptide 205-225 from the fusion protein of a group A strain. Peptide 205-225 (B) induced a proliferative T-cell response, demonstrating the existence of a T-cell epitope in this region of the fusion protein of group B viruses. Both peptides were able to induce a T-cell cross-reactive proliferation when mice were primed with either the homologous or the heterologous peptide. ELISA were performed using synthetic peptides or whole virus (from group A and B) as antigens. Mice anti-peptide sera recognized both homologous and heterologous peptides. A similar pattern was observed with RS virus strains. In indirect immunofluorescence assays, both anti-peptide rabbit sera recognized human nasal epithelial cells infected with A or B strains of RS virus. In contrast, while anti-peptide 205-225 rabbit serum from group A neutralized group A and B strains of RS virus, anti-peptide 205-225 rabbit serum from group B was unable to neutralize a group A virus, although it neutralized a group B strain. These results are similar to the immune response observed in children following primary RS virus infection.     

M proteins of group G streptococci: mechanisms of resistance to phagocytosis

Group G streptococci that express M protein and resist phagocytosis in human blood (virulent strains) were compared with strains of groups G and A that are readily phagocytosed (avirulent). Virulent group G streptococci were less effective (P < .05) as activators of the alternative complement pathway (ACP) than were avirulent streptococci. In immunofluorescence studies, C3 bound more avidly to avirulent than to virulent group G streptococci. Resistance of virulent group G strains to ACP opsonization and to phagocytosis was markedly diminished by removal with pepsin of the type-specific portion of the M molecule. Preincubation with fibrinogen did not diminish ACP activation or C3 binding by virulent group G and A streptococci but did exert an antiphagocytic effect. Given the similarity of M proteins of groups G and A in structure and function, other microbial constituents are likely responsible for differences in the spectra of illnesses attributable to the two serogroups.     

Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF

Apoptosis in vivo is followed almost inevitably by rapid uptake into adjacent phagocytic cells, a critical process in tissue remodeling, regulation of the immune response, or resolution of inflammation. Phagocytosis of apoptotic cells by macrophages has been suggested to be a quiet process that does not lead to production of inflammatory mediators. Here we show that phagocytosis of apoptotic neutrophils (in contrast to immunoglobulin G-opsonized apoptotic cells) actively inhibited the production of interleukin (IL)-1beta, IL-8, IL-10, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-alpha, as well as leukotriene C4 and thromboxane B2, by human monocyte-derived macrophages. In contrast, production of transforming growth factor (TGF)-beta1, prostaglandin E2, and platelet-activating factor (PAF) was increased. The latter appeared to be involved in the inhibition of proinflammatory cytokine production because addition of exogenous TGF-beta1, prostaglandin E2, or PAF resulted in inhibition of lipopolysaccharide-stimulated cytokine production. Furthermore, anti-TGF-beta antibody, indomethacin, or PAF receptor antagonists restored cytokine production in lipopolysaccharide-stimulated macrophages that had phagocytosed apoptotic cells. These results suggest that binding and/or phagocytosis of apoptotic cells induces active antiinflammatory or suppressive properties in human macrophages. Therefore, it is likely that resolution of inflammation depends not only on the removal of apoptotic cells but on active suppression of inflammatory mediator production. Disorders in either could result in chronic inflammatory diseases.     

A role for scavenger receptors in phagocytosis of protein-coated particles in rainbow trout head kidney macrophages

In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout (Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 microm in diameter), specifically coated with 125I labeled Atlantic salmon (Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.     

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.     

Mice deficient for the IL-3/GM-CSF/IL-5 beta receptors]

Candida species are increasingly important fungal pathogens. The reaction of rat alveolar macrophages (AM) to Candida albicans was compared with that to C. glabrata and C. krusei. Phagocytosis of C. glabrata was similar to that of C. albicans, but significantly slower for C. krusei due to reduced attachment. After opsonization, attachment of C. albicans and C. krusei to AM was significantly increased and there was no significant difference between the two species. The oxidative metabolism of AM with candida species was two to three times higher than that of the resting AM both during and 24 h after the phagocytosis. All three species showed a considerable fraction (5-10%) of phagolysosomes with pH > or = 6.5 after 3 h and a smaller percentage (1%) after 24 h.     

Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes

The objective of this study was to differentiate the roles of opsonins and phagocytic cells in the size-dependent hepatic uptake of liposomes in the submicron region. The extent of opsonization decreased with the decrease in size of liposomes (from 800 to 200 nm in diameter) and no enhancement of uptake was observed at 200 nm. There was no effect of liposome size on the uptake of unopsonized liposomes. Serum was pretreated with empty liposomes of each size and its opsonic activity was measured in the perfused liver. The small liposomes could not consume the opsonic activity, while the larger ones did so substantially. These results suggest that opsonins bind to liposomes depending on the size of liposomes and phagocytic cells take up liposomes in proportion to the extent of opsonization. Size-dependent liposome degradation in serum was also found, which was consistent with the size-dependent complement activation, because liposomes with this composition have been shown to be degraded by complement. The mechanism of opsonization was examined by treating serum at 56 degrees C for 30 min or with anti-C3 antiserum. Since both treatments inhibited the opsonic activity, the hepatic uptake of liposomes is considered to occur via complement receptor. In conclusion, the size of liposomes affected complement recognition, and the liposomes were taken up by the liver depending on the extent of opsonization.     

Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria

The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.     

Endogenous opioid tone in patients with rheumatoid arthritis

A monoclonal antibody giving a dominant reaction with the group-specific polysaccharide of streptococcus group B in an ELISA test has been developed. The purified polysaccharide exhibited a high positivity with reference anti B streptococcal antiserum in the ELISA test. Cross-tests of antibodies with other groups of streptococci provided a minimum cross-reaction only in the case of G streptococci. Monoclonal antibodies were prepared using Streptococcus agalactiae S 589 MT strain isolated from a case of bovine mastitis which does not express Ia, Ib, II, III, IV and V type antigens, nor C, R and X protein antigens.     

Simultaneous identification of antibodies to Brucella abortus and Staphylococcus aureus in milk samples by flow cytometry

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureus antibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortus or S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-microm-diameter latex particles. The difference in size between B. abortus and S. aureus permits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.     

In vitro phagocytosis of Escherichia coli and release of lipopolysaccharide by adhering hemocytes of the silkworm, Bombyx mori

A primary culture containing adhering hemocytes mainly granular cells from the silkworm, Bombyx mori, was used to investigate in vitro phagocytosis of Escherichia coli. Phagocytosis was confirmed to occur in this system by microscopic observation. Lipopolysaccharide (LPS) concentration in the culture medium was measured by a Limulus test and a higher LPS concentration was detected in phagocytosis-occurred samples than in control samples, which omitted either E. coli cells or adhering hemocytes. Moreover, it was found that LPS containing sample but not control samples strongly induces gene expression of cecropin B, an antibacterial protein. These results suggest that bacterial cell wall components like LPS released by phagocytosis play an important role in the induction of insect antibacterial proteins.     

Phagocytosis and killing of suspended and adhered bacteria by peritoneal cells after dialysis

OBJECTIVE: To determine the effect of dialysis fluid containing various glucose concentrations on the phagocytosis and killing of Staphylococcus aureus by rat peritoneal cells under conditions mimicking the in vivo situation. DESIGN: Phagocytosis and killing were evaluated by quantitation of the killing capacity of macrophages after in vivo phagocytosis of the bacteria as well as by an in vitro flow cytometric assay of the phagocytosis and killing of adhered bacteria by peritoneal cells. ANIMALS: Male Wistar rats. MAIN OUTCOME MEASURE: It was expected that the intraperitoneal administration of dialysis fluid would impair the capacity of peritoneal cells to eliminate bacteria. RESULTS: The first test revealed no effects of glucose concentration or dwell time on the killing of phagocytosed bacteria by macrophages, median percentages ranging between 29% and 64%. In the second series of experiments no effect of glucose concentration on the phagocytosis and killing of adhered bacteria was found either; however, longer dwell times significantly enhanced both the phagocytosis (at a dwell time of 1 hour, under 20%; at dwell times of 4 or 18 hours, above 20%, p < 0.02) and the killing (at a dwell time of 1 hour, under 53%; at dwell times of 4 and 18 hours, above 70%, p < 0.01). CONCLUSIONS: Glucose concentration has no effect on the phagocytosis and killing of Staphylococcus aureus, whereas the dwell time significantly enhances both of these functional capacities of peritoneal cells if the bacteria are adhered to surfaces.     

Mammalian transcobalamin II metabolism. The immunological and the biological cross-reactivity of mammalain transcobalamin II

Assay of the reactivity between the chicken anti-rabbit transcobalamin II antiserum and the sera of 19 vertebrate species was carried out by both immuno-diffusion and Sephadex G-200 gel filtration column chromatography. Mammalian transcobalamin II cross-reacted with the antiserum whereas the serum vitamin B-12 binders of the bird, amphibian reptile and fish did not. The biological activity of the purified rabbit transcobalamin II was assessed using reticulocytes or erythrocytes of human, rabbit, guinea pig and rat. The purified rabbit transcobalamin II promoted the uptake of vitamin B-12 by the cells but showed a great variation in its activity. It is suggested that the rabbit transcobalamin II is immunologically and biologically similar to the serum transcobalamin II of the mammalian species studied.     

Opsonizing activities of IgG, IgM antibodies and the C3b inactivator-cleaved third component of complement in macrophage phagocytosis

Phagocytosis of SRBC by guinea-pig peritoneal macrophages is enhanced by opsonizing IgG antibody alone. IgM antibody requires the presence of bound C3. Treatment of C3b coated SRBC with purified C3b inactivator (yielding EAIgM C1423d) does not reduce attachment to, and phagocytosis by, peritoneal macrophages. This finding suggests the existence of a C3d receptor on peritoneal macrophages. EC43b intermediates which have been produced by removing IgM antibody by mercaptoethanol treatment and by subsequent removal of C1 and C2, are phagocytosed despite the absence of IgM antibody. Furthermore, treatment of EC43b with C3b inactivator does not change phagocytosis. Thus, IgM antibody does not appear to be a necessary prerequisite for the stimulation of phagocytosis, C3b or C3d alone being sufficient.     

Characterization of variable immunodominant antigens of Cowdria ruminantium by ELISA and immunoblots

Cross-immunization experiments have revealed a significant antigenic diversity of the isolate of Cowdria ruminantium which needs to be characterized for the development of vaccines. We identified polymorphic immunodominant antigens by ELISA and immunoblot. Using serum from a goat immune to the Gardel stock of Cowdria (isolated in Guadeloupe) adsorbed on antigen of the Senegal stock of this pathogen, distinct serogroups were revealed by ELISA among six isolates from different geographical origins. Furthermore, a goat serum directed against the Senegal stock and adsorbed on Gardel antigens was shown to be specific for the Senegal stock, thus confirming the existence of serotypes in Cowdria. The Major Antigenic Protein 1 (MAP1) of Cowdria was shown to have variable antigenic determinants. Also in a group of variable proteins ranging from 23 to 29 kDa, one antigen of 26-27 kDa had a determinant specific for the Gardel isolate. These polymorphic antigens may be relevant components of Cowdria ruminantium for a vaccine as the sera revealing these antigens originated from a goal surviving a lethal challenge. However, the presence of T-cell epitopes and the ability of the these antigens to confer protection to ruminants remain to be investigated. The production of a rabbit antiserum against this group of polypeptides will be of great use for their purification and for the screening of expression libraries.     

Recognition and clearance of liposomes containing phosphatidylserine are mediated by serum opsonin

Liver uptake of liposomes containing phosphatidylserine was studied in a single pass liver perfusion system and found to be serum dependent. The effectiveness of serum in mediating liposome uptake by the liver depends on liposomes size. Large liposomes appeared to be opsonized more efficiently and, therefore, taken up more by the liver than the smaller ones. The effects of liposomes size on liver uptake did not occur in the absence of serum. Treatment of serum at 56 degrees C for 30 min abolished the serum activity, suggesting the involvement of complement components. Inhibition of the hemolytic activity of complement through the alternative pathway by PS-containing liposomes suggests that components in this pathway are responsible for liposome opsonization. Liposomes containing phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate compete in different degrees for serum components which mediate the liver uptake of PS-containing liposomes. These results suggest that the opsonization of liposomes by serum opsonins are the determining factors for the recognition and clearance of liposomes by the RES. Complement components are most likely involved in this process. The results presented here are relevant to the use of liposomes as drug delivery vehicle in vivo and to the PS-mediated clearance of red blood cells from the blood circulation.