Augmentation of activities of peripheral granulocytes and of resistance against bacterial infection after administration of G-CSF into mice]

We evaluated the metabolic capability of murine peripheral granulocytes after administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by quantitative flow cytometric assay for H2O2-dependent oxidative product formation. Intraperitoneal administration of a daily dose of 10 micrograms of rhG-CSF for 5 days induced doubling of the leukocyte population. Differential counting of peripheral leukocytes and scattergram by flow cytometry showed an increased mature granulocyte population. After stimulation with phorbol myristate acetate, the granulocytes of the rhG-CSF-administered mice demonstrated some hyperresponsive population and an increased H2O2 production. The hyperresponsive population showed H2O2 production 4-6 times higher than did normal cells. Granulocytes from the G-CSF-treated mice revealed an augmented phagocytic activity and an increased expression of Mac-1 molecules. Moreover, mice treated with G-CSF showed an enhanced resistance against intravenous infection with a lethal dose of E. coli. Granulocytes showing such markedly increased oxidative metabolism may be a significant component of the host defence to various infective organisms.     

Reticulo-Endothelial Activity in Mice Infected with Plasmodium vinckei

SYNOPSIS. The effect of P. vinckei infections on the phagocytic activity of the reticulo-endothelial system in mice has been investigated. Changes occurring in the pattern of R.E. activity show that there is slight stimulation of phagocytosis 24 hours after the initiation of the infection at which time parasites appear in the blood. The stimulation continues and reaches a peak on the 3rd day, following which it drops to subnormal levels by the 6th and 7th days when the animals die. The parasitaemia rises abruptly from the 4th day onwards, this increase bearing a direct relationship to the fall in the level of phagocytic activity. The changes which occur during this infection are compared with those previously reported for Plasmodium gallinaceum in chicks.     

Does ceftazidime or latamoxef affect the phagocytic capabilities of human granulocytes?

In comparative studies made in patients with pneumonia we have treated 14 cases with Latamoxef and 16 cases with Ceftazidime. We have focused attention on the side effects of these drugs and on the phagocytic capacity of granulocytes. In our patients we have noted a decrease of bactericidal activity of plasma and granulocytes and an elevated NBT reduction capability of resting granulocytes. The other tested function of the granulocytes exhibited only small variations. The applied therapy caused the bactericidal activity to be increased in granulocytes and plasma. Both drugs had no harmful effect on the phagocytic activities of granulocytes.     

Evaluation as a function of granulocytes in hyperimmunoglobulinemia E syndrome with recurrent infections

The hyper-IgE syndrome with recurrent infections (HIESRI) is characterized by skin and respiratory infections due to Staphylococcus aureus and several fungi infections which are frequently associated with tissue damage. A deficiency in the chemotaxis of phagocytic cells has been documented to explain these findings; however, the expression of adhesion molecules, the secretion of cytokines that activate granulocytes and the production of oxygen reactive molecules have not been evaluated in HIESRI. Six HIESRI patients were evaluated for the following parameters: (1) secretion of GM-CSF and IL-5 by mitogen and antigen-activated mononuclear cells, (2) the chemotactic response of FMLP-activated granulocytes, (3) the respiratory burst of PMA-activated granulocytes, and (4) the expression of L-selectin and CD11b in PMA-activated granulocytes. Human recombinant GM-CSF and culture supernatants were evaluated for capacity to modulate granulocytic function. Compared to controls, HIESRI patients showed a normal production of GM-CSF and an increase in the basal secretion of IL-5. No significant differences were observed for chemotaxis, respiratory burst or L-selectin and CD11b expression. The GM-CSF did not modulate these functions in granulocytes from HIESRI patients, but culture supernatants applied to granulocytes inhibited chemotaxis, increased respiratory burst and caused the shedding of L-selectin from the granulocyte surface. The 6 HIESRI patients were nonsymptomatic during the time of this research due to a program of continued treatment; findings suggest that granulocytes are activated more easily in response to proinflammatory factors and that production of these factors is higher in HIESRI.     

Effect of stress on viral-bacterial synergy in bovine respiratory disease: novel mechanisms to regulate inflammation

The severity of bovine respiratory infections has been linked to a variety of factors, including environmental and nutritional changes, transportation, and social reorganization of weaned calves. Fatal respiratory infections, however, usually occur when a primary viral infection compromises host defences and enhances the severity of a secondary bacterial infection. This viral-bacterial synergy can occur by a number of different mechanisms and disease challenge models have been developed to analyse host responses during these respiratory infections. A primary bovine herpesvirus-1 (BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolytica results in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model to demonstrate that stress significantly altered the viral-bacterial synergy resulting in fatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-like receptors (TLR) expression and increased pro-inflammatory responses which contribute to the severity of a Mannheimia haemolytica infection. TLRs play a critical role in detecting bacterial infections and inducing pro-inflammatory responses. It is difficult to understand, however, how stress-induced corticosteroids could enhance this form of viral-bacterial synergy. Nuclear translocation of the glucocorticoid receptor activates cell signalling pathways which inhibit both TLR signalling and pro-inflammatory responses. The apparent conundrum between stress-induced corticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

Natural killer cells vs cytotoxic T cells in the peripheral blood of virus-infected mice

The cell-mediated immune response of mice against various enveloped RNA and DNA viruses expressed by immune lymphocytes from the spleen and the peripheral blood (PBL) were compared. PBL from mice of various strains infected with vaccinia virus, vesicular stomatitis virus (VSV), or lymphocytic choriomeningitis virus (LCMV) were tested on histocompatible or incompatible target cells infected with the homologous virus. PBL from immune mice showed clear H-2 restriction, but additionally, they expressed high natural killing (NK) activity on YAC-1 cells. The high NK-cytolytic activity of PBL on YAC-1 differed significantly from that expressed by splenic lymphocytes. In both lymphocyte populations lysis was detected as early as 1 day after infection; NK activity decreased in the spleen after day 4 post infection, whereas that of PBL persisted at high levels for up to 10 days after infection. Treatment of mice with anti-asialo GM1 in vivo abrogated NK activity in PBL effector cells tested in vitro. These results may explain some of the difficulties to observe MHC-restricted cytotoxic T cells in PBL from humans or primates during primary infections with virus.     

Schistosoma mansoni: extramedullar eosinophil myelopoiesis induced by intraperitoneal glass implants in chronically infected mice

Intraperitoneal glass implants in mice with chronic Schistosoma mansoni infections induce intense local myeloid reactions involving essentially myeloid granulocytes. The same phenomenon is not observed in normal mice nor in mice with acute schistosomal infection. An association of myelopoiesis with differentiated macrophages mobilized on glass implants and with dense ameboid cells located inside myeloid foci was detected. A macrophage-dependent induction of this eosinophil reaction is postulated.     

Influence of HIV-infection on the phagocytic activity of monocytes/macrophages and granulocytes

Because of the great importance of phagocytosis as a key process in host defence, the influence of HIV-infection on the phagocytic activity of monocytes/macrophages (M0/MAC) and granulocytes was investigated. Therefore, blood samples from the peripheral blood of 70 HIV-infected individuals were incubated with fluorescein isothiocyanate (FITC) labeled Escherichia coli. The uptake of the bacteria was monitored by flow cytometer analysis. A strong and significant increase in the relative number of phagocytic granulocytes was observed ranging from 12.8% in an uninfected control collective to over 30% in AIDS patients. This effect was obtained for all patients and independent of the stage of disease. For monocytes, only marginal changes were found in their phagocytic function. These data suggest that the high susceptibility of HIV patients for secondary infections is not linked to a loss of phagocytic ability of monocytes/macrophages and/or granulocytes.     

Nippostrongylus brasiliensis: mast cells and histamine levels in tissues of infected and normal rats.

The role of the spleen during Babesia microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendothelial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci.     

Effect of the influenza A virus on the sensitivity of mice to infection caused by Streptococcus group B

Study of the capacity of group B streptococci for causing the development of infection in mice has revealed the virulence of the cultures for mice to be determined by the serovar of the streptococcus, the infective dose, and the amount of type-specific polysaccharide. Under the conditions of mixed viral-bacterial infection, influenza A virus was shown to influence the development of bacterial infection in the animals in two ways: to increase the virulence of an avirulent strain and to decrease the pathogenicity of a virulent one in streptococcal monoinfections. Simultaneously with viral infection, the stimulation of the multiplication of an avirulent strain in the lungs of mice was observed, while in the control groups of the animals the elimination of bacteria from the lungs was registered. No additional accumulation of the infective virus in the lungs of mice in the presence of streptococci was found.     

Activation of rainbow trout, Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin

The chemiluminescent response of kidney phagocytic cells was significantly increased by the oral administration of bovine lactoferrin to rainbow trout, Oncorhynchus mykiss. The phagocytic activity of kidney cells was also increased by administration of bovine lactoferrin to the fish. Activation of kidney cells was also observed in vitro with cells cultured in the presence of bovine lactoferrin.     

Modulation of phagocytic activity in cultured sertoli cells

Phagocytic activity of rat Sertoli cells that were cultured in vitro has been evaluated as the ability to internalize polystyrene beads. Our data demonstrate that these ceils are active phagocytes and that such phagocytic activity is under negative control by follicle-stimulating hormone (FSH). The hormonal control is mediated by increased intracellular levels of cAMP. Moreover Sertoli cdl responds to tuftsin, an oligopeptide known to act only on macrophages and granulocytes, by increasing up to five times its phagocytic activity. Phagocytic uptake of polystyrene beads is associated with dramatic changes of the cellular shape. Such morphological modifications are significantly reduced under FSH stimulation. The physiological implications of the data are discussed.     

Stimulation of hematopoiesis and antibacterial resistance by interleukin-1

Summary The beneficial effects of IL-1 and other cytokines on hematopoiesis and on resistance to infection are profound. IL-1 stimulates proliferation of bone marrow cells in normal mice and potentiates the recovery of peripheral blood neutrophils in mice with drug-induced neutropenia. Prophylactic cytokine administration provides an elevated level of natural resistance to infections which is correlated with increased numbers of phagocytic leukocytes. These studies suggest that IL-1 has potential clinical application as a therapy to limit bone marrow dysfunction and immuno-suppression and to augment hematopoiesis and natural immunity. Further research will continue to elucidate the mechanisms whereby interleukins and colony-stimulating factors act, and interact, to promote restoration of leukocyte production and to enhance host resistance.     

Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa

Chronic inflammation of the lung, as a consequence of persistent bacterial infections by several opportunistic pathogens represents the main cause of mortality and morbidity in cystic fibrosis (CF) patients. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes, additionally, mature macrophages from different tissues, although having a number of common activities, exhibit variation in some molecular and cellular functions. In order to highlight possible intrinsic macrophage defects due to CFTR dysfunction, we have focused our attention on in vitro differentiated macrophages from human peripheral blood monocytes. Here we report on the contribution of CFTR in the bactericidal activity against Pseudomonas aeruginosa of monocyte derived human macrophages. At first, by real time PCR, immunofluorescence and patch clamp recordings we demonstrated that CFTR is expressed and is mainly localized to surface plasma membranes of human monocyte derived macrophages (MDM) where it acts as a cAMP-dependent chloride channel. Next, we evaluated the bactericidal activity of P. aeruginosa infected macrophages from healthy donors and CF patients by antibiotic protection assays. Our results demonstrate that control and CF macrophages do not differ in the phagocytic activity when infected with P. aeruginosa. Rather, although a reduction of intracellular live bacteria was detected in both non-CF and CF cells, the percentage of surviving bacteria was significantly higher in CF cells. These findings further support the role of CFTR in the fundamental functions of innate immune cells including eradication of bacterial infections by macrophages.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

The natural cytotoxicity receptor 1 contribution to early clearance of Streptococcus pneumoniae and to natural killer-macrophage cross talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligand(high) lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-ligand(dull) macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.     

Human anti-Pseudomonas aeruginosa outer membrane proteins IgG cross-protective against infection with heterologous immunotype strains of P. aeruginosa.

In order to develop an effective means to treat and prevent Pseudomonas aeruginosa infections, we have purified P. aeruginosa outer membrane protein (Oprs)-specific human IgG antibody using a large-scale affinity column. In this study, we investigated the cross-protective activity of the purified anti-Oprs IgG against various immunotype strains of P. aeruginosa. The anti-Oprs IgG reacted with Oprs isolated from seven Fisher-Devlin immunotype strains of P. aeruginosa and was able to promote opsonophagocytic killing of all seven immunotype strains by human phagocytic cells. Administration of 500 microg anti-Oprs IgG to mice raised the LD50 of the P. aeruginosa strains by 8-250-fold, indicating the protective capacity against heterologous P. aeruginosa strains as well as homologous strains. In contrast, despite high titers against P. (aeruginosa Oprs, total serum IgG isolated from burn patient sera was no better than normal serum IgG in protecting mice from infection with P. aeruginosa. These data demonstrate that the affinity-purified human anti-Oprs IgG could afford protection against heterologous immunotype P. aeruginosa strains and provide a rationale to use anti-Oprs IgG as an adjunct for treatment of P. aeruginosa infections in humans.     

Lymphocyte subpopulations and level of the proinflammatory cytokines in the blood of patients with hepatitis C and with combined variant of hepatitis C and B

Subpopulation of cytotoxic lymphocytes CD8 having both secretory and cytolytic antiviral activity is supposed to play the essential role in the virus elimination. Inflammatory reactions are also of importance in the hepatitis C pathogenesis, their intensity being regulated by anti-inflammatory cytokins. This study was aimed at determination of lymphocyte subpopulation indices--the relative content of cells carrying markers CD4, CD8, CD16, CD19, CD72, the parameters of the phagocytic activity of granulocytes and monocytes, as well as serum concentrations of anti-inflammatory cytokines IL-1, IL-6 and TNF-alpha. These indices were evaluated in a group 132 patients with confirmed hepatitis C or mixed hepatitis B + C diagnosis, depending on the disease form and markers of the infectious process activity (as determined in the PCR test for hepatitis C virus RNA) in comparison with a group of healthy donors. In patients with variant hepatitis under study a growth in anti-inflammatory mediators concentration was observed along with decreased indices of lymphocyte subpopulations responsible for cell-mediated immunity and the phagocytosis parameters. In the case of mixed hepatitis these differences were shown to be more manifested.