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PubMed2001

Are low-density granulocytes the major target cells of classical swine fever virus in the peripheral blood?

The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post-infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low- and high-density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post-infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high-density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus-positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.

Animals↗

PubMed1987

The third component of complement is transcribed and secreted by cultured human endothelial cells.

Monotypic cultures of passaged human umbilical vein endothelial cells secrete C3 protein, which is specifically immunoprecipitable from cell lysates and conditioned medium. A transcript migrating at 5.3 kilobases hybridizes with a mouse-derived C3 DNA probe in parallel cultures of endothelial cells, hepatoma cells (HepG2), and freshly isolated human monocytes. Steady-state transcript levels were as follows: HepG2 cells, 1.0; monocytes, 0.05; endothelial cells, 0.01. These data suggest that C3, an indispensible component for activation of complement by both the classical and alternative pathways, is locally synthesized in the vascular bed.

Cells, Cultured↗

PubMed2003

[The "classical" macrophage marker CD68 is strongly expressed in primary human fibroblasts].

AIM: Monoclonal antibodies against the human homologue of mouse macrosialin, CD68, are generally commercialized as markers for human monocytes and macrophages. Indeed, CD68 is considered as a selective marker for human myeloid cells, although several previous immunohistochemical studies indicate that some antibody clones also react with other hematopoietic and non-hematopoietic cell types. The aim of our study was to verify these observations and to evaluate the reliability of CD68 as a macrophage marker. METHODS: We investigated protein and RNA expression of CD68 in various fibroblast types and carcinoma cell lines as compared to monocytes and macrophages using immunohistochemistry, flow cytometry, and specific RT-PCR. Different monoclonal antibody clones against CD68 were applied including KP-1 and EBM11. RESULTS: As expected, the intensity of immunohistochemical and flow cytometric CD68 staining was dependent on both the antibody clone and the fixation procedure. However, fibroblasts isolated from normal skin, normal breast, breast tumor tissue, and osteoarthritis synovia clearly expressed CD68 protein at levels comparable to macrophages. The specificity of CD68 expression in fibroblasts was verified by RT-PCR which also showed some tumor cell types to express CD68 mRNA. CONCLUSION: Our findings clearly demonstrate that the expression of CD68 is not restricted to the macrophage lineage. This is highly relevant for experimental and diagnostic purposes, since anti CD68 antibodies cannot be accepted without reservations for the discrimination of myeloid cells and fibroblasts even in paraffin sections after formalin fixation.

Antigens, CD↗

PubMed1991

Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages.

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.

Calcium↗

PubMed2000

Toxic effects of SLE serum on normal monocytes in vitro: cell death induced by apoptosis related to complement dysfunction.

The aim of this study was to assess toxic effects of systemic lupus erythematosus (SLE) serum on blood peripheral mononuclear cells from healthy donors and to evaluate if complement activation was involved. Monocytes from a healthy donor were incubated with 20 sera from ten SLE patients in both high and low disease activity states. After incubation non-adherent cells were analysed by flow cytometry. Serum from six SLE patients induced an increased cell death, four in active disease only, one in the inactive state and one in the active and the inactive state. Five of these sera, three with high and two with low disease activity, induced an increased apoptosis in the monocytes. Proportion of apoptotic cells correlated inversely with C1q and C3 concentration in the active disease sera, but not with disease activity as evaluated by SLEDAI. Apoptosis could be induced by addition of active C1s or antigen/antibody complexes to normal serum before incubation. Serum with complexes added induced increased tumour necrosis factor-alpha secretion from mononuclear cells, but SLE patient sera did not. The results demonstrate that the toxic effect of serum from SLE patients on healthy monocytes is explained by induction of apoptosis. The induction process is suggested to be connected with complement activation in the serum.

Adolescent↗

PubMed1983

Inhibition of complement-dependent phagocytosis by monocytes following pre-incubation with immune complexes and interaction with plasma.

Normal monocytes have been pre-incubated with soluble immune complexes (IC) (BSA-anti-BSA) or heat-aggregated human IgG (AHG) (ICs) at pathophysiological concentrations. If these 'IC-monocytes' are allowed to interact with plasma they show reduced phagocytosis of Candida guilliermondii targets. Under the pre-incubation conditions used, 2% of offered ICs remain cell associated and their binding to monocytes is energy-independent and complement-independent. At these low concentrations of ICs, only C3-dependent phagocytosis is inhibited, although Fc-dependent phagocytosis is inhibited at high concentrations of AHG. Inhibition of C3-dependent phagocytosis requires a critical concentration of plasma (greater than or equal to 2%), complement C3, and functionally intact classical and alternative complement pathways. The observations could be explained by blockade or modulation of C3 receptors by IC associated C3b or by an effect on ingestion. The abnormality induced in normal monocytes in vitro closely resembles that found in monocytes from patients with active IC disease.

Antigen-Antibody Complex↗

PubMedDate not supplied

[Interleukin-1 production in patients with subacute and chronic opisthorchiasis].

Interleukin-1 (IL-1) production by peripheral blood lymphocytes was studied in 3 patients with the subacute and in 28 patients with the chronic stage of the disease. Monocyte culture was obtained by the H. R. Recalde method, IL-1 activity was evaluated by the S. B. Mizel modification of the classic LAF-ASSAY. In the subacute stage of the disease IL-1 activity was 3.3 fold lower that in healthy controls and lower than in the total group of patients with chronic opisthorchiasis (Kact. 0.64 +/- 0.09, 2.12 +/- 0.17, 1.83 +/- 0.11, respectively, p < 0.01, p < 0.01). In those with the syndrome of chronic gastroduodenitis IL-1 production was lower than in patients with the syndrome of cholepathy (Kact. 1.55 +/- 0.18 and 2.03 +/- 0.16, p < 0.01). Immediately after praziquantel therapy IL-1 production fell from Kact. 1.82 +/- 0.13 to 1.18 +/- 0.06 (p < 0.01), but 5-6 months later the production of the cytokine normalized in convalescents.

Acute Disease↗

PubMed2006

Does HLA-B27 influence the monocyte inflammatory response to lipopolysaccharide?

OBJECTIVES: How human leucocyte antigen B27 (HLA-B27) contributes towards arthritis susceptibility is still unclear, but effects on the response to bacteria unrelated to the classical antigen presenting role of B27 have been suggested. This study investigated whether HLA-B27 modulates the innate response to lipopolysaccharide (LPS), a component shared between all Gram negative bacteria that can trigger reactive arthritis. METHODS: Pools of U937 transfectants expressing either HLA-B27, HLA-A2 or the expression plasmid alone were differentiated with phorbol 12-myristate 13-acetate and stimulated with LPS. Supernatants were analysed for tumour necrosis factor-alpha (TNF-alpha) secretion and the gene expression profiles of unstimulated and LPS-stimulated cells were determined by microarray analysis. Changes in gene expression that are indicative of an unfolded protein response (UPR) were also analysed by quantitative polymerase chain reaction (PCR). RESULTS: TNF-alpha secretion, a biological marker of the inflammatory response to LPS, was not significantly different between U937-B27 and U937-control. No differences in gene expression between unstimulated U937-B27 and U937-control lines were detected. Both U937-control and U937-B27 exhibited a stereotypic response to LPS. Only one gene, OAS2, was differentially expressed by these cell lines, and this was confirmed by quantitative PCR. Analysis of XBP-1 splicing suggested that the UPR is induced following the LPS stimulation, but this increase was seen in all transfectants. CONCLUSIONS: The expression of B27 does not profoundly alter the gene expression following LPS stimulation. Therefore, additional signals, such as those provided by cytokines or intracellular infection, may be required to reveal any influence of B27 expression on the inflammatory response.

Gene Expression Profiling↗

PubMed1995

Peripheral blood and synovial fluid monocyte expression of interleukin 1 alpha and 1 beta during active rheumatoid arthritis.

OBJECTIVE: To investigate the mononuclear cell (MNC) types from peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) expressing interleukin-1 beta and -1 alpha (IL-1 beta, IL-1 alpha). METHODS: PB and SF MNC from 16 patients with classical or definite RA and controls were analyzed by flow cytometry for IL-1 beta and IL-1 alpha staining. RESULTS: MNC from normal individuals do not stain for either IL-1 beta or IL-1 alpha. By contrast, PB and SF CD14+ monocytes from patients with active RA show significant staining for surface and intracellular IL-1 beta and IL-1 alpha. Furthermore, the erythrocyte sedimentation rate (ESR) correlates with the surface expression of IL-1 alpha (r = 0.52, p < 0.05) while the number of active joints (i.e., tender and/or swollen) correlate with the intracellular expression of IL-1 beta (r = 0.58, p < 0.03) in CD14+ monocytes. CONCLUSION: The analysis of IL-1 beta and IL-1 alpha expression on CD14+ monocytes from peripheral blood by flow cytometry could be a potential immunological marker of disease activity in RA and its variation could be used to monitor the effects of therapy.

Antigens, CD↗

PubMed1982

[Streptococcal A-induced polyclonal T-cell activation leads to a secondary functional activation of human B-cells].

Human lymphocytes are activated by cell walls or cytoplasma membranes of A-streptococci. Even in health human volunteers lymphocyte proliferation, lymphokine production and immunoglobulin synthesis is induced by high concentrations of streptococcal cell components following a pattern of polyclonal lymphocyte activation. We were interested in the special cellular requirements in this particular model. In cell separation and restitution experiments the observed B-cell activation was identified as the consequence of a primary monocyte-dependent polyclonal T-cell activation. Furthermore, lymphocyte proliferation and lymphokine production proved to be a monocyte- and T-cell-dependent process as well. Thus, lymphocyte activation induced by somatic streptococcal structures in humans depends upon a pattern of cell cooperation of mononuclear subpopulations well-known in a series of classical mitogen-induced lymphocyte activation.

Antigens, Bacterial↗

PubMed1987

Modulation of FcR function by complement: subcomponent C1q enhances the phagocytosis of IgG-opsonized targets by human monocytes and culture-derived macrophages.

We have investigated the interaction of C1q, a subunit of the first component of complement, with human monocytes and culture-derived macrophages. Adherence of these mononuclear phagocytes to surfaces coated with C1q induced a marked enhancement of the phagocytosis of sheep erythrocytes opsonized with IgG anti-Forssman antibody (EA-IgG). This C1q-mediated enhancement of phagocytosis was dose dependent, and was specifically blocked by pretreatment of the C1q-coated surfaces with F(ab')2 anti-C1q. The augmentation of FcR-mediated phagocytosis by C1q was determined to be a result of the interaction between the C1q and the phagocytic effector cell, and was not due to interaction between the surface-bound C1q and the EA-IgG. Neither resting nor N-formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes were induced by C1q to increase FcR-mediated phagocytosis. Experiments conducted with purified fragments of C1q suggest that the C1q phagocytosis enhancement signal resides in the collagen-like tail domain of the molecule. This region is the same portion of the molecule previously shown to interact with the cell surface C1q receptor. Native type I collagen was unable to enhance FcR-mediated phagocytosis by mononuclear phagocytes. It has been demonstrated that C1q can be localized to areas of inflammation, and additionally C1q can be secreted by macrophages in culture. In view of these findings and the results of our present study, we hypothesize that C1q could provide local, direct, and non-opsonic enhancement of phagocytosis by mononuclear phagocytes in areas of infection and inflammation.

Cell Adhesion↗

PubMed2003

A phylogenetic comparison between acute monocytic leukemia cells and monocytes-macrophages in lower vertebrates.

In humans, monocytes and macrophages (Mphi) play a central role in immune regulation, tissue maintenance and pathogen control. In lower vertebrates, a few studies have been conducted on Mphi like cells. In acute monocytic leukemia monocytic cells, as immature cells restrained in one of the phases of their ontogenesis, would offer the opportunity to rebuild an archaic condition helpful to understand the phylogenesis. Therefore, aim of this work was to characterize in the Rainbow trout (Salmo Gairdneri Richardson) Mphi and compare them with acute leukemia monocytic cells. In the trout, Mphi's morphology is similar to that of mammals. In particular, Mphi possess an irregular embryoshaped nucleus occupying 2/3 of the cell, while the peripheral cytoplasmic profile is irregular with extroflexed plasmalemma and pseudopods. A morphological transition towards Mphi is featured by a wavy hyaline classical membrane and an irregular and extroflexed surface. Some aspects of erythrophagocytosis represented a finding of great interest indicating that the hemocatheretic function could take place directly in circulation. This condition, also observed in human acute monocytic leukemia, suggests that the information to the erythrophagocytosis is restrained under physiological conditions. Non-specific esterases, which are positive in human Mphi smear and Mphi from human lymph node tissue, were also positive in the teleost studied but with a dysomogeneous pattern. Consequently non-specific esterase system is phylogenetically conserved. A lack of immune-reactivity with the anti-CD68 monoclonal antibody (MoAb) on smear and trout tissue sections was observed. On the contrary, strong positivity was detected on human lymph node sections. In trout, the presence of Mphi and circulating Mphi like cells exhibiting an erythrocatheretic function in the circulation would indicate a primordial function that has later been replaced by the liver and the spleen.

Animals↗

PubMed2003

Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease.

Current knowledge states that periodontal diseases are chronic inflammatory reactions raised in response to periodontopathogens. Many cell types and mediators, including Th1 and Th2 lymphocytes, cytokines and chemokines, appear to be involved in the immunopathogenesis of periodontal diseases. Chemokines, a family of chemotactic cytokines, bind to specific receptors and selectively attract different cell subsets to the inflammatory site. They can also interact with classical cytokines and modulate the local immune response. In order to study the role of chemokines in periodontal diseases, we examined the expression of chemokines, chemokine receptors and cytokines by means of reverse transcription-polymerase chain reaction (RT-PCR) techniques. Characteristic patterns of such factors' expression were found in gingival biopsies from patients presenting with aggressive periodontitis and chronic periodontitis. The expression of the chemokines macrophage inflammatory protein-1 alpha (MIP-1alpha) and interferon-gamma inducible protein 10 (IP-10) and of their respective receptors, CCR5 and CXCR3, were more prevalent and higher in aggressive periodontitis, and associated with higher interferon-gamma (IFN-gamma) expression and lower interleukin-10 (IL-10) expression. In contrast, chronic periodontitis patients exhibited a more frequent and higher expression of monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR4, and higher expression of IL-10. It is possible that chemokines, in addition to the classical cytokines, are involved in the immunopathogenesis of periodontal disease, driving the migration and the maintenance of several inflammatory cell types such as polymorphonuclear leukocytes, dendritic cells (DCs), natural killer cells, macrophages, and subsets of lymphocytes in the gingival tissues. These cells are thought to participate in the inflammatory and immune reaction that takes place in periodontal disease, killing pathogens, presenting antigens, and producing cytokines. The selective recruitment of polarized lymphocyte subsets could result in differential cytokine production at the site of response, which is supposed to determine the stable or progressive nature of the lesion. Besides, the role of chemokines as activators and chemoattracts of osteclasts may be involved in the determination of disease severity.

Adult↗

PubMed2007

Bleomycin treatment causes enhancement of virus replication in the lungs of SHIV-infected macaques.

Pneumonia is a major complication of human immunodeficiency virus (HIV) pathogenesis but it develops only after prolonged infection. We used the macaque model to explore a hypothesis that the disease is a two-stage process, the first stage being establishment of the viral infection in the lung and the second being amplification of virus replication by host factors induced by chemical agents or opportunistic pathogens in the lung. Bleomycin, a chemical known to induce diffuse alveolar damage and pulmonary fibrosis with accumulation of macrophages and a rich T helper type 2 (Th2) cytokine environment, was inoculated intratracheally into five of eight SHIV 89.6P-infected macaques and into one uninfected macaque. Three additional simian HIV (SHIV)-infected macaques without bleomycin treatment served as untreated virus controls. Although none of the animals became clinically ill, bleomycin induced classical host responses in the lungs of all the treated, virus-infected macaques. There was enhanced production of the chemokine, monocyte chemotactic protein-1 (MCP-1), that had previously been shown to cause enhanced replication of the virus. Four of the five treated animals developed more productive SHIV infection in the lungs compared with the infected untreated animals. Enhanced virus replication was found primarily in infiltrating macrophages. Enhanced replication of the virus in the lungs was associated with host factors induced by the drug and supported the hypothesis for a two-stage process of pulmonary pathogenesis.

Animals↗

PubMed1999

Complement activation by mycoloyl glycolipids from Mycobacterium tuberculosis and Rhodococcus ruber.

In this study, we examined complement activation by mycoloyl glycolipids (MGL) such as trehalose-6,6'-dimycolate (TDM), often termed cord factor, and trehalose-6-monomycolate from Mycobacterium tuberculosis and Rhodococcus ruber, and also examined the effect of complement binding to MGL on phagocytosis by human monocytes. TDM and TMM, but not glucose mycolate, mannose mycolate or fructose mycolate which differ from TMM only in carbohydrate moiety, exhibited complement activation. TDM and TMM of M.tuberculosis exhibited stronger complement activation than those of R.ruber, the mycolic acids of which are much shorter than those of M.tuberculosis. Neither mycolic acids nor trehalose, which are products of TDM and TMM by hydrolytic cleavage, exhibited no complement activation. TDM activated complement through the alternative pathway, since supplementation with C4-deficient serum completely restored classical pathway-mediated hemolytic activity of complement which had been previously consumed by TDM. Next, we examined the effects of TDM and TMM on phagocytosis by human monocytes. Coating of heat-killed Staphylococcus aureus cells with TDM or TMM did not enhance their phagocytosis by monocytes, while, in the presence of complement, phagocytosis of these cells increased significantly. These findings suggest that TDM and TMM act as virulence factors that enhance the entry of mycobacteria into phagocytes via binding of C3 through activation of the alternative complement pathway.

Adjuvants, Immunologic↗

PubMed1983

Complement-mediated binding of monocytes to intermediate filaments in vitro.

An in vitro model for complement (C)-mediated monocyte binding to injured cells is described. This model is based on the property of cytoskeletal intermediate filaments (IMF) to directly and independently activate serum C via the classic pathway. Fixed monolayers of cultured fibroblasts, endothelial cells, and L 132 epithelial cells were each used as targets. The authors first subjected the target cells to limited detergent extraction in order to make permeable the plasma membrane, and then exposed them to normal human serum. Immunohistologic analysis demonstrated that this process permitted binding of clearly demonstrable amounts of C1q, C4, and C3 to cytoplasmic loci containing IMF. The target cells were then incubated for various lengths of time with peripheral blood mononuclear cells containing about 20% monocytes. Preferential binding of monocytes was demonstrated by histochemical and immunologic staining methods. Only 5% of the attached cells were B lymphocytes. By transmission electron microscopy the attached cells exhibited typical monocyte morphology. Attachment to target cells involved contact with areas rich in IMFs and led to signs of activation; both an increased number of cytoplasmic organelles and phagocytosis of target cell material were observed. Attachment of monocytes was clearly dependent on serum treatment of the targets; the ratio was 10:1 when compared with control experiments using target cells that had not been incubated with serum. It is reasonable to consider that bound monocyte-derived macrophages associate with activated C components, most likely C3b fragments or C1q, for which monocyte surface receptors have been established. This mechanism may be triggered in various inflammatory reactions involving cell and tissue injury and as an event in the final stages of macrophage-mediated removal of injured or nonviable cells.

Animals↗

PubMed1987

Formation of the membrane attack complex of complement (MAC) on erythrocytes from monocyte-produced terminal complement components.

By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.

Animals↗

PubMed2002

A CD18-dependent protein kinase C beta-mediated alternative cell death pathway of activated monocytes.

Activated monocytes become resistant to numerous death stimuli including death receptors. Given that the uncontrolled activation of monocytes/macrophages and their persistence can lead to severe inflammatory conditions, it is critical to define the pathways that control their elimination. We previously reported that ligation of HLA-DR molecules on peripheral blood-derived monocytes induces their death. To investigate the mechanisms of HLA-DR-mediated death in monocytes, we used the THP-1 monocytic cell line as a model. We show that while THP-1 are equally resistant to HLA-DR- and to Fas-mediated death, treatment of THP-1 with IFN-gamma renders them sensitive to HLA-DR- but not to Fas-mediated death. Both activation of the Src family protein tyrosine kinase and classical protein kinase C (PKC) occur through HLA-DR, but only PKC activation is involved in HLA-DR-mediated death of these cells. Moreover, HLA-DR-mediated cell death of activated monocytes implicates a regulatory loop between the HLA-DR/CD18 complex and the downstream activation of PKCbeta. Thus, our study identifies an alternative physiological signaling pathway of monocyte death, and further investigation on its regulation is likely to provide significant insights into the control of monocyte homeostasis and inflammation.

CD18 Antigens↗