Role of specific antibody in interaction of leptospires with human monocytes and monocyte-derived macrophages

It has previously been shown that human neutrophils ingest and kill nonpathogenic Leptospira biflexa in the absence of serum but that pathogenic Leptospira interrogans is not ingested by neutrophils even in the presence of normal serum. We extended this study by examining the interactions of human monocytes and monocyte-derived macrophages with pathogenic L. interrogans (serovar icterohaemorrhagiae) and evaluating the opsonizing effect of serotype-specific immune serum on the phagocytosis of pathogenic leptospires by monocytes and neutrophils. Leptospires were incubated with monocytes in pellets at 37 degrees C in 5% CO2. No ingestion or killing of pathogenic leptospires occurred when 10% normal serum was used. However, when the pathogenic leptospires were pretreated with serotype-specific immune serum, monocytes or neutrophils in pellets ingested 96% of the organisms and killed 94% of those ingested. Microscopic observations of the interaction confirmed that phagocytosis of the opsonized pathogenic leptospires by monocytes, monocyte-derived macrophages, and neutrophils had occurred. The opsonizing effect of specific antibody may play an important role in the mechanism of host defense against leptospirosis.     

Opsonins in normal mouse serum for the phagocytic killing of Proteus mirabilis by murine neutrophils.

An assay of phagocytic killing by murine neutrophils in homologous serum was used to determine the nature of the opsonins in normal mouse serum for phagocytic killing of Proteus mirabilis. Leucocytes from the peritoneal cavities of mice given an intraperitoneal inoculation of brain-heart infusion broth 3 h previously, phagocytosed and killed P. mirabilis in a 2-h assay in the presence of 10% serum from normal mice. The serum factors supporting phagocytic killing were heat-labile (50 degrees C or 56 degrees C for 30 min) and could be absorbed at 37 degrees C but not 4 degrees C by three different species of Gram-negative bacteria. The tested species of Gram-positive bacterium did not absorb the activity. At the end of the assays, greater than 90% of leucocyte-associated bacteria were associated with neutrophils. Leucocytes from unstimulated peritoneal cavities (less than I% neutrophils) did not kill bacteria in this assay, in contrast to leucocyte suspensions containing up to 98% neutrophils. These findings indicated that the phagocytic killing of P. mirabilis in this assay was mediated by neutrophils, and that complement fixation by the alternative pathway provided necessary opsonins in normal mouse serum.     

Killing ofTaenia hydatigena oncospheres by sheep neutrophils

Neutrophils collected from the mammary glands of uninfected sheep or from sheep infected withTaenia hydatigena, attached to and killedT. hydatigena oncospheres in vitro in the presence of serum from infected sheep. Infected sheep serum alone was not deleterious to the parasite in vitro. Fc receptors for antibody were detected on both normal and immune neutrophils; they were present at a greater density on the latter. Immune neutrophils were more reactive towards oncospheres than normal neutrophils and formed extensive capsules around the parasite. Fc receptors were not detected on oncospheres. It is hypothesised that neutrophils may kill the parasite by producing hydrogen peroxide and the superoxide anion, both of which are toxic to a variety of cell types and protozoa. The function of antibody may be to facilitate attachment of neutrophils to oncospheres by way of their Fc receptors.     

Opsonins in normal mouse serum for the phagocytic killing of Proteus mirabilis by murine neutrophils

An assay of phagocytic killing by murine neutrophils in homologous serum was used to determine the nature of the opsonins in normal mouse serum for phagocytic killing of Proteus mirabilis. Leucocytes from the peritoneal cavities of mice given an intraperitoneal inoculation of brain-heart infusion broth 3 h previously, phagocytosed and killed P. mirabilis in a 2-h assay in the presence of 10% serum from normal mice. The serum factors supporting phagocytic killing were heat-labile (50 degrees C or 56 degrees C for 30 min) and could be absorbed at 37 degrees C but not 4 degrees C by three different species of Gram-negative bacteria. The tested species of Gram-positive bacterium did not absorb the activity. At the end of the assays, greater than 90% of leucocyte-associated bacteria were associated with neutrophils. Leucocytes from unstimulated peritoneal cavities (less than I% neutrophils) did not kill bacteria in this assay, in contrast to leucocyte suspensions containing up to 98% neutrophils. These findings indicated that the phagocytic killing of P. mirabilis in this assay was mediated by neutrophils, and that complement fixation by the alternative pathway provided necessary opsonins in normal mouse serum.     

Functional activity of individual abscess neutrophils from mice.

In the absence of antibiotic therapy, viable bacteria can persist within intra-abdominal abscesses in mice for at least 10 weeks. The mechanisms contributing to this survival are unknown, but abscess-derived neutrophils have impaired abilities to kill, in vitro, organisms engulfed in vivo. In order to determine whether subpopulations of abscess neutrophils might be discernible on the basis of phenotypic or functional criteria, cells from murine intra-abdominal abscesses were examined for phagocytic activity, CR3 expression, and H2O2 production in response to soluble and particulate stimuli. With respect to phagocytosis of Proteus mirabilis, abscess cells were no less efficient than peritoneal exudate neutrophils; no significant subpopulation of cells was incapable of phagocytosis in the presence of normal mouse serum. Using flow cytometry to examine abscess neutrophils for CR3 expression, we found that no subpopulations of cells were observed with unstimulated cells or with cells incubated with either phorbol 12-myristate 13-acetate or bacteria and serum. Intracellular H2O2 levels were measured by using the probe 2',7'-dichlorofluorescin diacetate. In general, incubation with phorbol 12-myristate 13-acetate resulted in similar increases in H2O2 production in all cells of the population. However, stimulation with bacteria and serum revealed a variable but consistent, poorly responsive subpopulation of neutrophils in abscess cell populations. Cell-sorting experiments showed that cells from the poorly responsive section of the FACS profile contained significantly higher numbers of abscess-derived bacteria, suggesting the presence of a subpopulation of viable abscess neutrophils harboring persisting viable bacteria.     

Neutrophil activity in abscess-bearing mice: comparative studies with neutrophils isolated from peripheral blood, elicited peritoneal exudates, and abscesses.

Intraabdominal abscesses were induced in mice by intraperitoneal inoculation of Bacteroides fragilis and Escherichia coli plus bran as the abscess-potentiating agent. Six- or seven-day-old abscesses were mechanically disaggregated in buffer, and the cells obtained were fractionated on discontinuous Percoll density gradients. Neutrophil populations of different density, each approximately 90% pure, were isolated. When the abscess-derived neutrophils were subsequently incubated with normal serum in vitro under aerobic conditions, the viability of the gram-negative bacteria that had been phagocytosed within the abscess did not change significantly. This anergy to intracellular bacteria (on subsequent incubation in vitro under optimal conditions for phagocytic killing) was also found for neutrophils that had been obtained from abscesses induced by a mixture that included Proteus mirabilis plus B. fragilis and from those induced by E. coli plus P. mirabilis. While unable to significantly kill intracellular organisms that had been phagocytosed in vivo, the abscess-derived neutrophils could engulf and kill organisms to which they were exposed in vitro. Neutrophils from abscesses induced by P. mirabilis only plus bran killed that organism introduced in vitro significantly more effectively than the organisms that had been engulfed in vivo. In contrast, neutrophils from abscesses induced by the gram-positive organism Staphylococcus aureus plus bran were able to kill their intracellular organisms on subsequent incubation in vitro as effectively as they could kill added S. aureus. Neutrophils isolated from the peripheral blood and from induced peritoneal exudates of abscess-bearing mice were able to phagocytose and kill organisms in vitro with greater efficiency than abscess-derived neutrophils. The mechanism whereby neutrophils from abscesses induced by the gram-positive organism S. aureus can kill the organisms phagocytosed in vivo on subsequent in vitro incubation, in contrast to the relative anergy to their intracellular organisms displayed by neutrophils derived from abscesses induced by combinations of gram-negative bacteria, is not known.     

Human neutrophils susceptibility to Paracoccidioides brasiliensis: an ultrastructural and cytochemical assay.

Paracoccidioidomycosis, caused by the dimorphic fungus Paracoccidioides brasiliensis, is the most prevalent systemic mycosis of Latin America, with Brazil accounting for 80% of the reported cases. The great number of neutrophils found in P. brasiliensis granulomas demonstrates the importance of polymorphonuclear neutrophils (PMN) cells during this mycotic infection. It has been found that neutrophils from healthy human donors can ingest and kill the fungus through a typical phagocytic process. The present work tests the phagocytic ability of neutrophils collected from patients that had had and were considered cured of paracoccidioidomycosis. Transmission electron microscopy and cytochemical studies indicate that patients' neutrophils eventually degenerate during phagocytosis of P. brasiliensis. Endogen peroxidase and NAD(P)H-oxidase are activated during the process showing that the respiratory burst and the neutrophil degranulation are triggered by the attachment of the yeast cells. Apparently these processes are not enough to kill P. brasiliensis. Although fungicidal activity can be determined by colony forming unit (CFU) counting, qualitative data suggest, as noted, that neutrophils from patients with treated paracoccidioidomycosis degenerate during the phagocytosis process. Hence, this work demonstrates the existence of a functional neutrophil deficiency against P. brasiliensis in susceptible individuals. The exact origin of this susceptibility is still to be determined in further studies.     

Neutrophil function in chronic liver disease

Neutrophil locomotion, phagocytosis and killing of Candida albicans, and plasma opsonization of brewer's yeast were studied in 44 out-patients with chronic liver disease. There were four diagnostic groups: alcoholic liver disease (ALD), chronic active hepatitis (CAH), primary biliary cirrhosis (PBC) and cryptogenic cirrhosis (CC). Results were compared with a control group of patients with non-malignant disorders of the upper alimentary tract. Neutrophil locomotion induced by zymosan-activated autologous plasma was significantly depressed in patients with ALD and, to a lesser extent, in cryptogenic cirrhosis. With plasma from healthy donors, the patients' neutrophils showed normal locomotion. Plasma from patients with CAH gave slightly but significantly reduced phagocytosis of both Candida albicans and brewer's yeast, but the patients' cells had normal phagocytic and killing activity in the presence of normal plasma. Thus, no intrinsic abnormality in neutrophil function was found in these patients, but plasma defects, which differed in cirrhoses of different underlying aetiology, led to impaired neutrophil locomotion or phagocytosis. No correlations were found between these plasma defects and circulating levels of C3, C4, immune complexes or IgA.     

A neutrophil disorder induced by capnocytophaga, a dental micro-organism.

We recovered capnocytophaga, a gram-negative anaerobe implicated in the pathogenesis of periodontal disease, from two patients with a history of dental infections. Neutrophils from both patients failed to acquire the asymmetric shape characteristic of normal neutrophils. Fluorescein staining of the patients' living neutrophils remained diffuse and patchy instead of showing the normal pattern in which the fluorescence is swept into the rear of the cell. The locomotion of one patient's neutrophils in vitro was less than 50 per cent of that of normal neutrophils, and migration of this patient's neutrophils into dermal abrasions was reduced, although phagocytosis and nitroblue tetrazolium reduction were normal. All abnormalities of neutrophil morphology and function disappeared after eradication of the capnocytophaga infections. Sonicates and culture medium of capnocytophaga contained a dialyzable substance that caused normal neutrophils to behave like neutrophils obtained from the infected patients.     

Neutrophil expression of tumour necrosis factor receptors (TNF-R) and of activation markers (CD11b, CD43, CD63) in rheumatoid arthritis.

In vitro analysis of polymorphonuclear neutrophils (PMN) has allowed various stages of cell activation to be distinguished, characterized by the expression level of specific membrane markers and of functional receptors. Among those, TNF-alpha receptors (TNF-R) are modulated by various PMN activators, a mechanism which may be important to control cell responses to TNF in inflammatory reactions such as rheumatoid arthritis (RA). PMN, isolated from the blood of 36 RA patients and from the synovial fluid of 23 of them, were analysed for membrane expression of the two TNF-R (p55 and p75). Soluble p55 and p75 (sTNF-R) and TNF concentrations were measured in the plasma and synovial fluid by specific ELISA assays. Our results show that PMN from the blood of RA patients bear a normal number of TNF-R, with a normal p55/p75 ratio, compared with PMN from normal controls. Soluble TNF-R levels were similar in patients and normal plasma. In spite of high endogenous TNF concentration, patients' circulating PMN were not activated, as shown by a CD11b/CD18 expression similar to that of control resting cells. In contrast with blood neutrophils, PMN from RA patients' synovial fluids had an activated phenotype, characterized by increased expression of CD11b, decreased expression of leukosialin, CD43, and the appearance on the plasma membrane of an azurophil granule protein, CD63. High levels of soluble TNF-R were measured in RA synovial fluids. Nevertheless, membrane TNF-R levels and p55 and p75 proportions were similar to those of PMN from normal blood. These results suggest the existence of regulatory mechanisms which maintain a stable neutrophil expression of TNF-R as well as a balance between both types of receptors in inflammatory situations where neutrophils are strongly activated.     

Role of stimulated neutrophils from patients with systemic lupus erythematosus in tissue injury,with special reference to serum factors and increased active oxygen species generated by neutrophils

To examine the possible correlation between tissue injury and neutrophil produced active oxygen (AO) species in patients with systemic lupus erythematosus (SLE), we studied the capacity of the serum from six patients with untreated, active SLE to generate AO and release lysosomal enzymes by normal neutrophils. Cultured endothelial cells from human umbilical cord vein were incubated with serum-stimulated neutrophils to assess AO-induced tissue injury. Serum from patients with bacterial infections and healthy individuals served as controls. AO production was highest in the neutrophils stimulated with SLE patient-derived serum, while lysosomal enzyme release was only slightly increased. SLE neutrophils with or without stimulation and SLE serum-stimulated normal neutrophils produced significantly high levels of cytotoxicity upon coincubation with⁵¹Cr-labeled human endothelial cells. These excessive cytotoxicities were reversed by the presence of superoxide dismutase and catalase, indicating the specificity of the AO effect on endothelial cell damage. These findings suggest that tissue damage in SLE may be partially due to excessive production of AO and that both neutrophils themselves and a serum factor which activates neutrophils are involved in the mechanism for vascular injury.     

Activated phenotype in neutrophils and monocytes from patients with primary proliferative polycythaemia.

AIM--To investigate whether monocytes and neutrophils from patients with primary proliferative polycythaemia (PPP) exhibit increased expression of markers of cell activation and, if so, whether they are associated with the phagocytic activity of these cells and concentrations of circulating cytokines. METHODS--Expression of CD11b, CD14, CD18, and CD64 on monocytes and neutrophils was assessed by flow cytometry. Phagocytosis was analysed using immunoglobulin opsonised Escherichia coli. Serum concentrations of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) were determined by bioassays, and interferon-gamma (IFN-gamma) by enzyme linked immunosorbent assay (ELISA). RESULTS--Patients with PPP (n = 18), when compared with normal subjects (n = 10), had increased percentages of CD64+ monocytes (52% v 36%) and neutrophils (42% v 11%) and of CD14+ neutrophils (36% v 18%). Monocytes from patients with PPP exhibited increased expression of CD64 (47 v 26) and of CD11b (65 v 36). These abnormalities were not found in patients with secondary (n = 8) or apparent (n = 13) polycythaemia. The percentage of neutrophils undergoing phagocytosis was higher in patients with PPP (mean 64%; n = 6) than in normal subjects (mean 42%; n = 5). G-CSF, GM-CSF and IFN-gamma concentrations in patients' serum samples were comparable with normal; M-CSF was not detected in any of the samples. There was no correlation between cytokine concentrations and the expression of CD11b, CD14, CD18, and CD64 on patients' phagocytes. CONCLUSIONS--Increased expression of CD11b and CD64 by monocytes, increased percentages of CD14+ and CD64+ neutrophils and the high phagocytic activity of neutrophils suggests that these cells are activated in vivo in patients with PPP. The phenotypic changes of PPP phagocytes were not associated with increased concentrations of circulating cytokines and probably reflect intrinsic abnormalities within the neoplastic PPP clone.     

Neutrophil function in workers exposed to organophosphate and carbamate insecticides.

Neutrophil function in 40 workers occupationally exposed to carbamate and organophophate insecticides were examined and compared to those of non-exposed individuals. Phagocytosis and intracellular killing of Candida albicans and Candida pseudotropicalis by neutrophils were studied. Two species of Candida were used since in individuals with myeloperoxidase deficiency neutrophils are unable to kill Candida albicans, while Candida pseudotropicalis can be effectively lysed. Phagocytosis of both antigens was normal in all the workers studied. On the other hand, there was a considerable reduction in the ability of neutrophils from exposed workers to kill Candida albicans whereas Candida pseudotropicalis was effectively lysed. This finding indicates some interference with the myeloperoxidase activity in the exposed population. The levels of cholinesterase activity in all workers were normal. These results demonstrate that exposure to carbamates and organophophates insecticides may lead to changes in neutrophil function even in workers presenting no impairment in the cholinesterase activity.     

Defective activation of neutrophils after splenectomy.

Neutrophil chemotaxis and phagocytosis in the presence of serum from 20 patients who had undergone splenectomy and from 15 healthy volunteers was studied. The mean distance migrated by normal neutrophils in the presence of serum from the patients after splenectomy was significantly less than that when normal serum was used (p less than 0.005). The percentage of neutrophils phagocytosing a yeast was also significantly reduced in the presence of serum from patients after splenectomy (p less than 0.02). In addition, when neutrophils from these patients were studied both chemotaxis and phagocytosis were enhanced in normal compared with autologous serum (p less than 0.05).     

Phagocytosis and intracellular killing of the contagious equine metritis organism by equine neutrophils in serum.

Equine neutrophils were combined with Haemophilus equigenitalis (contagious equine metritis organism; CEMO) or Escherichia coli in low- and high-antibody-titer serum to evaluate the neutrophils ability to phagocytize and kill these bacteria. More E. coli than CEMO were phagocytized at each time period. After 120 min in low-antibody-titer serum, 56.3% of the E. coli and 34.3% of the CEMO were phagocytized. A total of 45% of CEMO and 74.9% of E. coli were phagocytized by 120 min when neutrophils were in high-antibody-titer serum. More than 75% of the ingested E. coli and 90% of the ingested CEMO were killed within 210 min of incubation. Fewer E. coli than CEMO were killed at any given time period. Ultrastructural examination showed CEMO to be degraded in the neutrophil. Degradation was the most extensive in neutrophils in high-titer serum. It is suggested that CEMO is a pathogenic extracellular bacterium incapable of prolonged intracellular survival and that it is slower to be phagocytized than a nonpathogenic E. coli.     

Bactericidal activity of serum from cystic fibrosis patients for Pseudomonas aeruginosa

The bactericidal action of serum from 61 adult patients with cystic fibrosis (CF) against autologous and heterologous strains of Pseudomonas aeruginosa has been studied. CF serum had a similar bactericidal action to normal human serum (NHS) against a reference panel of strains. Six CF sera had a selective inability to kill autologous strains of pseudomonas, which were sensitive to NHS and to sera from other CF patients. The six sera had normal levels of complement and immunoglobulin and were bactericidal to other strains. A titratable blocking factor was present in these sera and it interfered with the bactericidal action of NHS on the appropriate strain. This factor was present in the IgG-containing fractions of serum obtained by ion-exchange chromatography, but was not removed from the serum by absorption with the pseudomonas strain. Some CF sera may fail to kill sensitive strains of pseudomonas because of the development of a blocking IgG antibody against naturally occurring bactericidal IgM antibody.     

Localization on encapsulated Cryptococcus neoformans of serum components opsonic for phagocytosis by macrophages and neutrophils.

Previous studies have shown that the cryptococcal capsule inhibits phagocytosis of Cryptococcus neoformans by macrophages and neutrophils. In this study, the binding sites of potential serum opsonins in immune and nonimmune sera were determined by immunoelectron microscopy, and the results were compared with the results of phagocytosis of the yeasts by mouse peritoneal macrophages and human neutrophils. Immunoglobulin G (IgG) from normal human serum showed low-density binding at the capsular surface and at sites throughout the capsule. Complement component C3 from normal serum bound heavily at the capsular surface. IgG from rabbit capsular antiserum showed relatively dense deposition at the capsular surface and at sites throughout the capsule. Cells opsonized with heat-inactivated human serum were engulfed poorly by both macrophages and neutrophils, indicating that the low-density deposition of IgG produced by normal serum was not adequate for opsonization. Yeasts opsonized with normal human serum were engulfed in large numbers by neutrophils and to a lesser extent by macrophages, indicating that neutrophils in particular were able to effectively utilize the opsonically active C3 which normal human serum deposited at the capsular surface. Yeasts opsonized with rabbit anticapsular serum were engulfed by both macrophages and neutrophils, indicating that the high density of surface IgG produced by capsular antiserum is an effective opsonin for both cells. These results suggest that the complement-neutrophil system is a possible defense mechanism in the nonimmune host.     

Mechanisms of resistance against experimental Trypanosoma cruzi infection. Requirements for cellular destruction of circulating forms of T. cruzi in human and murine in vitro systems.

The sensitivity of host (circulating) forms of T. cruzi to cell-mediated immunological destruction and requirements for the reaction were examined. Both human and mouse leucocytes were found to kill significant numbers of parasites in the presence of specific antibodies against the flagellates. Antibody involvement was confirmed by the marked ihhibitory effects on cytotoxicity that resulted from the addition to reaction mixtures of either aggregated normal IgG or purified protein A. Similar inhibition was observed when antiserum to T. cruzi was pre-absorbed with an insoluble protein A preparation. In addition, immunoglobulins present in normal mouse serum failed to support cytotoxicity by cells with demonstrated effector capacity in parallel antibody-containing reactions. In this system, human peripheral blood lymphocytes, neutrophils and eosinophils but not adherent mononuclear cells were able to kill T. cruzi. Also active were mouse lymphoid cells, neutrophils and adherent mononuclear cells. Minimal effector:target cell ratios resulting in detectable trypanosome killing were 0 x 2 and 0 x 6 for human and mouse lymphoid cells, respectively. The present results are relevant to the understanding of the possible mechanisms underlying the protective effects of the immune response against T. cruzi infection.     

Lymphocyte blastogenesis in nephrotic syndrome.

Peripheral blood lymphocytes from children with steroid-responsive nephrotic syndrome showed inhibition of the blastogenic response to PHA-P when incubated in the presence of autologous serum. Inhibition was also evident when patients' cells were incubated with normal human serum or when normal cells were incubated with patients' serum. This inhibition was not seen when the patients were in remission. In experimental nephrosis in rats induced by a single injection of the aminonucleoside of puromycin, a similar pattern of lymphoblastogenic inhibition was seen. Thus human steroid-responsive nephrotic syndrome and experimental nephrosis both show similar disturbances of lymphocyte responsiveness to mitogenic stimulation.     

Polymorphonuclear activation in leprosy. I. Spontaneous and endotoxin-stimulated reduction of nitroblue tetrazolium: effects of serum and plasma on endotoxin-induced activation.

Spontaneous nitroblue tetrazolium (NBT) reduction was evaluated in neutrophils from patients with the different types and forms of leprosy, and compared with reduction obtained form cells from normal controls. Leucocytes from the same subjects were stimulated in vitro by endotoxin, and the rise in percentage of cells reducing NBT was determined. Patients of all groups, with the exception of those with reactional lepromatous leprosy (RLL) had an essentially normal proportion of reducing cells. Neutrophils were normally activated by endotoxin. This indicates that while Mycobacterium leprae does not by itself stimulate leucocytes from leprosy patients, there is no overall anergy of neutrophils in lepromatous or other forms of leprosy. In RLL the proportion of reducing cells was significantly raised. Stimulation with endotoxin was able further to enhance this proportion, but not above levels reached by stimulation of normal cells. Neutrophil activation could not be reproduced by mixing serum from highly activated RLL patients with normal leucocytes. An inhibitory effect of serum and plasma over in vitro endotoxin activation of neutrophils was found.