Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae

Gonococcal porins (Por) from strains FA19 (Por-1, serogroup A), MS11 (Por-2, serogroup B) and FA6434 (Por-5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non-denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole-cell radioimmunoprecipitation and were broadly cross-reactive within serogroups. Post-immune, but not pre-immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outermembrane protein Rmp.     

High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess

To better understand the role of capsular polysaccharide (CPS) K1 or K2 in Klebsiella pneumoniae liver abscess as well as the development of metastasis to eye, neutrophil phagocytosis of 70 CPS isolates including K1 (n = 23)/K2 (n = 10), non-K1/K2 (n = 37) was evaluated by flow cytometry, fluorescence imaging, and electron microscopy. K1/K2 isolates were significantly more resistant to phagocytosis (P < 0.0001) than non-K1/K2 isolates and displayed increased resistance to intracellular killing. Although mucoid phenotype (M-type) K1/K2 isolates were significantly more resistant to phagocytosis (P = 0.0029) than M-type non-K1/K2, no significant difference in the phagocytosis rate was observed between K1/K2 isolates with M-type and non-M-type (P = 0.0924). Mucoidy is an associated factor that was predominant in K1/K2 isolates, but which itself is not an independent influence on phagocytic resistance. The K1/K2 CPS proved significantly more resistant to phagocytosis than non-K1/K2 CPS in liver abscess isolates (P < 0.0001) and non-abscess isolates (P = 0.0001), suggesting that K1/K2 isolates were generally more virulent in both liver abscess and in non-liver abscess conditions. These findings indicate that resistance of CPS K1 or K2 K. pneumoniae to phagocytosis and intracellular killing presumably contributes to their high prevalence in liver abscess and uniquely in endophthalmitis.     

A new type of anti-ganglioside antibodies present in neurological patients

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.     

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein detected in children with cystic fibrosis inhibit neutrophil-mediated killing of Pseudomonas aeruginosa

Antineutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) were repeatedly found in cystic fibrosis (CF) patients. We analyzed the effect of BPI-ANCA in inhibiting neutrophil-mediated killing of Pseudomonas aeruginosa. The bactericidal effect expressed as percentage of killed bacteria after 1 h incubation with neutrophils was 55% when the neutrophils were pretreated with normal human serum, ranged from 49 to 63% with the sera from control BPI-ANCA-negative groups and sharply decreased to the mean 30.5% (range 8-51%) in the presence of BPI-ANCA. Furthermore, the effect mediated by BPI-ANCA was dose dependent and reflected the titer of BPI-ANCA in tested sera.     

Soluble factors from rabbit spleen cells kill and lyse Treponema pallidum in vitro

Antibody and complement immobilize (kill) Treponema pallidum in vitro. Recent evidence also documents immobilization by soluble factors released by activated macrophages and lymphocytes. Immune-mediated lysis of treponemes, however, has not been reported. The findings in this paper focus on apparent treponemal lysis by rabbit splenic cell preparations. Using cells from animals infected testicularly for 9 to 12 days, unfractionated splenic preparations, as well as adherent and nonadherent preparations, killed and lysed T. pallidum. Phagocytosis alone could not explain the detrimental effects of adherent cells. When cytochalasin B was used to block phagocytosis, decreases in treponemal numbers were still detected. In related studies, immune rabbit sera did not enhance treponemicidal activity of the adherent cells. To assess the specificity of these reactions, T. pallidum was incubated with two monocyte-like cell lines (human U937 and mouse P388D1). Neither cell line was detrimental, and treponemal numbers were not lowered. The soluble nature of the treponemicidal factors from adherent and nonadherent preparations was shown by physically separating these cells from the organisms and demonstrating treponemal killing and lysis. In summary, clearance of T. pallidum from infected tissues is probably at least partially attributed to macrophage phagocytosis. Our findings suggest another mechanism involving lytic factors secreted by activated adherent and nonadherent cells.     

In vitro effect of actinomycin D on human neutrophil function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.     

The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces

Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae-induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.     

The effect of cytokines on bactericidal activity of murine neutrophils

Abstract A range of recombinant cytokines have now been shown to modify aspects of the phenotype and function of human and murine neutrophils. However, few reports describe modification of the bactericidal activity of neutrophils. We therefore examined the recombinant murine cytokines tumor necrosis factor-α (TNF-α, 10–1000 ng ml⁻¹) and granulocyte macrophage-colony stimulating factor (GM-CSF, 10–1000 U ml⁻¹) for their ability to increase the bacterial killing capacity of murine neutrophils. Neutrophils from either bone marrow (fresh or cultured), or peritoneal exudates, or abscesses, were pre-incubated with either cytokine for 30–60 min and the killing of Proteus mirabilis, Escherichia coli , or Bacteriodes fragilis was examined in the presence or absence of serum over a 90 min period. Only for one combination was a small but significantly enhanced level of bacterial killing observed, the phagocytic killing of P. mirabilis by peritoneal exudate neutrophils in the presence of GM-CSF and serum. With this exception there was no enhancement of bacterial killing for the range of combinations of neutrophils and bacterial species tested. In contrast, at the concentrations tested for effect on bactericidal activity, TNF-α and GM-CSF were able to significantly upregulate CR3(but not FcγRII) expression on mouse neutrophils. There results indicate that upregulation of CR3 as an index of neutrophil activation does not necessarily correlate with increased bactericidal activity.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Endothelial cells potentiate phagocytic killing by macrophages via platelet-activating factor release

The immunomodulatory function of endothelial cells (EC) includes the initiation of leukocyte margination, diapedesis, and activation through the upregulation of various cell surface-associated molecules. However, the effect that EC have on the phagocytic function of neighboring monocytes and macrophages is less well described. To address this issue, microvascular EC were cocultured with murine peritoneal macrophages, first in direct contact, then in a noncontact coculture system, and macrophage phagocytosis and phagocytic killing were assessed. The presence of increasing concentrations of EC resulted in a dose-dependent increase in macrophage phagocytic killing. This stimulatory effect was inhibited in a dose-dependent manner by the pretreatment of macrophage/EC cocultures with WEB-2086 or CV-6209, specific platelet-activating factor (PAF)-receptor antagonists, but not by anti-tumor necrosis factor-alpha, anti-interleukin (IL)-1alpha, or anti-IL-1beta. Furthermore, the effect was reproduced in the absence of EC by the exogenous administration of nanomolar concentrations of PAF. Microvascular EC potentiate macrophage phagocytic killing via the release of a soluble signal; PAF appears to be an important component of that signal.     

Inhibition of normal rat macrophage functions by soluble tumor products

Summary The phagocytic and chemotactic activities of normal rat peritoneal macrophages were inhibited by sera from tumor-bearing rats (TBR) and 3 M KCl extracts of tumor mass. However, sera from Corynebacterium parvum- or Listeria monocytogenes-treated TBR did not inhibit phagocytosis. On the other hand, sera from C. parvum-treated, but not from L. monocytogenes-treated TBR still inhibited the chemotactic response of the normal macrophages. Furthermore, 3 M KCl extracts of tumors from C. parvum-treated TBR did not inhibit phagocytosis and chemotactic response of the same cells. Similar results were obtained with extracts of tumor masses from L. monocytogenes-treated rats. It is suggested that treatment with bacterial immunomodulators can influence the release from neoplastic cells of soluble products influencing normal macrophage functions.     

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.     

Partial myeloperoxidase deficiency in preleukemia

In seven subjects with partial and apparently acquired form of myeloperoxidase (MPO) deficiency, some functional properties of neutrophils (PMNs) were studied. Five patients suffered from preleukemia, one from diabetes mellitus and one from carcinoma of the breast with bone marrow metastases. Intracellular bactericidal activity, oxygen consumption and superoxide radical production were within normal limits. In three patients with preleukemia, the serum opsonic activity was markedly reduced (less than m-3SD) in an autologous system, but normal in the presence of pooled normal serum. Decreased opsonic activity was also found when these patient's sera were assayed in the presence of normal PMNs. Since the levels of IgG and C3 were comparable in the patients' sera and the pooled serum, a deficiency of another unknown opsonin or the presence of an opsonization inhibitor has to be postulated. The partial MPO defect apparently doesn't decrease the intracellular killing of Staphylococcus aureus by PMNs. The known susceptibility to bacterial infections in preleukemia may be explained by the reduction of serum opsonization conducing to a secondary decrease of the ingestion and killing of bacteria by the PMNs.     

The role of grancalcin in adhesion of neutrophils

Grancalcin is a protein specifically expressed in neutrophils and monocytes/macrophages. The function of grancalcin has not been identified. Grancalcin-deficient neutrophils were previously demonstrated to exert normal recruitment to the inflamed site, NADPH oxidase activation, extracellular release of secondary granules, apoptosis and activation-induced Ca2+ flux. In this study we analyzed granule numbers in resting and activated grancalcin-deficient neutrophils, their phagocytic activity and adherence to extracellular matrix proteins. Results revealed normal phagocytosis and degranulation of grancalcin-deficient neutrophils, while their adhesion to fibronectin was decreased by 60%. Consistently, the processes associated with neutrophil adhesion, such as formation of focal adhesion complexes and spreading, were also impaired in grancalcin-deficient neutrophils by 89 and 38%, respectively. In contrast, adherence to other extracellular matrix proteins: collagen, laminin and vitronectin, was not significantly altered. We thus report for the first time a function of grancalcin.     

Anti-Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro

In this study, we demonstrated the anti-chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)- and weakly (W)-encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H- < W-strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan-activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan-activated serum was incubated with heat-killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O-acetyl groups in their molecules by ¹H-nuclear magnetic resonance spectroscopy. The de-O-acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de-O-acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti-chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O-acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.     

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.     

Plasmodium yoelii: antibody response in resistant and susceptible mouse strains

The numbers of antigen-reactive antibody-secreting cells, levels of parasite antigen-specific serum antibodies and numbers of red blood cells staining positive for surface immunoglobulin were determined for susceptible and resistant mouse strains following infection with Plasmodium yoelii 17x. As a control, these parameters also were measured using antigen prepared from normal red blood cells. The relatively susceptible C57BL/6 mice produced more antigen-specific antibody-secreting cells and had higher levels of immunoglobulin positive red blood cells than did DBA/2 mice, but the DBA/2 mice had more antigen-specific IgG in their sera. Both mouse strains possessed cells secreting antibody reactive with soluble normal red blood cell antigen; however, C57BL/6 mice had more IgG positive unparasitized RBC than did DBA/2 mice. Despite possessing fewer antibody positive normal RBC, DBA/2 mice had significantly higher levels of serum antibodies that reacted with soluble red blood cell antigen. These data indicate that levels of serum antibody may not reflect the amounts of antibody produced and that use of any single assay to assess the magnitude of the antibody response may give rise to misleading results.     

Functional capacity of neutrophils from class III obese patients

Class III obesity is associated with chronic inflammation and a variety of changes in immune function. Yet surprisingly little was known about the status of neutrophils that represent the first line of immune defense. The aim of this study was to assess key functions of neutrophils from class III obese patients, namely phagocytosis, superoxide production, chemotaxis, and response to endotoxin challenge, and compare their responses with lean controls. Thirty obese patients (BMI 48.8 ± 6.6 kg/m(2)) with comorbidities such as diabetes, hyperlipidemia, high blood pressure, etc. and nine lean (BMI between 20 and 25) subjects were enrolled in the study. Neutrophils from class III obese patients phagocytosed Escherichia coli (E. coli) at similar rates and with adequate numbers of bacteria taken up per cell compared with cells from lean subjects. Neutrophil production of superoxide, which is key to rapid killing of pathogens, showed modest diminution in the class III obese, which increased among patients with BMI >50. Chemotactic activity of neutrophils from class III obese patients was not altered. However, neutrophils from obese subjects showed an increased response to low-dose endotoxin, with concomitant reduced apoptosis and extension of their half-life compared with lean subjects, which suggests possible hyperresponsiveness of these neutrophils. Overall, neutrophil activity was not significantly altered by age, gender, diabetic status, or hyperlipidemia. Collectively, these results suggest that class III obese patients, even with comorbidities, have normal or nearly normal phagocytic, chemotactic, and superoxide generating capacity.     

Surface translocation of pactolus is induced by cell activation and death, but is not required for neutrophil migration and function

Pactolus is a cell surface protein expressed by murine neutrophils. Pactolus is similar to the beta integrins, except it lacks a functional metal ion-dependent adhesion site domain and is expressed without an alpha-chain partner. The majority of the Pactolus protein is held within the cell in dense granules in a highly glycosylated form. This intracellular form of Pactolus can be released to the cell surface following inflammatory activation or ligation of Pactolus on the cell surface. In addition, intracellular Pactolus translocates to the neutrophil surface following induction of apoptosis. Neutrophil activation studies suggest that Pactolus does not serve as an activating or phagocytic receptor for the neutrophil. To further define the function of Pactolus, a Pactolus-null mouse was generated. Pactolus-deficient animals mature appropriately and possess normal numbers of neutrophils, display appropriate migration into sites of inflammation, and combat introduced infections efficiently. These data suggest that Pactolus does not function as a neutrophil phagocytic or adhesion receptor, but may instead serve as a sugar-bearing ligand for lectin recognition by other cells.