German cockroach extract induces activation of human eosinophils to release cytotoxic inflammatory mediators

BACKGROUND: Eosinophils play an important role in the pathogenesis of allergic diseases. Sensitization and exposure to cockroach allergen have been demonstrated to be one of the major risk factors for the development of bronchial asthma. However, little is known regarding the functional capacity of cockroach extract antigen to activate human eosinophils. OBJECTIVE: We investigated whether German cockroach extract can activate human eosinophils to release cytotoxic inflammatory mediators. METHODS: Purified eosinophils from the peripheral blood were incubated with various concentrations (0-200 microg/ml) of German cockroach extract antigen. Effector functions of eosinophils were checked by degranulation and superoxide anion production. In addition, we examined surface expression of CD11b and CD69, and intracellular activation of p38 mitogen-activated protein kinase (p38 MAP kinase) in cockroach-stimulated eosinophils. RESULTS: German cockroach extract induced degranulation and superoxide production from human eosinophils. In addition, incubation of eosinophils for 3 h with the cockroach extract resulted in an increased level of the surface expression of CD11b and CD69. Furthermore, cockroach-induced superoxide production from eosinophils was significantly inhibited by the pretreatment of cells with a p38 MAP kinase inhibitor SB202190. Indeed, a large amount of phosphorylated forms of p38 MAP kinase was detected in cockroach-stimulated eosinophils. CONCLUSIONS: Our results suggest that German cockroach extract induces activation of human eosinophils to release cytotoxic inflammatory mediators such as superoxide and granular proteins.     

A purified polysaccharide isolated from Candida albicans induces antibody response in vitro by human peripheral blood lymphocytes and discriminates between sera from normal and Candida albicans-infected individuals

A purified polysaccharide extracted from Candida albicans (MPPS), stimulates in vitro synthesis of specific antibodies by human peripheral blood lymphocytes. These antibodies can be detected by a sensitive enzyme-linked immunoassay. The same assay can be applied to the quantitation of anti-candida antibodies in serum. Statistically significant differences were found between sera of normal and candida-infected individuals.     

Suppressive effect of anti-rheumatic drugs on interleukin-1 beta release from human peripheral blood monocytes

We developed an ELISA system for human IL-1 alpha and -beta release from silica-stimulated monocytes from healthy volunteers and tested the effect of several anti-rheumatic drugs including nonsteroidal anti-inflammatory drug (Ibuprofen). Anti-rheumatic drugs including Auranofin and Sulphasalazine suppressed IL-1 beta release significantly at therapeutic concentrations, whereas Bucillamine, Lobenzarit, D-Penicillamine and Ibuprofen did not. These results suggest a possible immunotherapeutic effectiveness of some anti-rheumatic drugs on rheumatoid arthritis through their inhibition of IL-1 beta release.     

Bacterial lipopolysaccharide induces release of tumor necrosis factor-alpha from bovine peripheral blood monocytes and alveolar macrophages in vitro

In this study, we demonstrate that freshly adherent bovine monocytes release tumor necrosis factor-alpha (TNF-alpha) in response to stimulation with bacterial lipopolysaccharide (LPS). TNF-alpha was detected using actinomycin D-treated WEHI-164 murine fibrosarcoma cells as targets in an 18 hr cytotoxicity assay. Doses of LPS from 20 ng/ml to 20 micrograms/ml were capable of inducing bovine TNF-alpha. The kinetics of TNF-alpha release from bovine monocytes demonstrated peak levels of cytotoxic activity at 1-3 hr post-LPS treatment, with a subsequent decline to background levels by 18 hr post-LPS treatment. A monoclonal antibody that neutralizes recombinant human TNF-alpha (rHuTNF-alpha) significantly reduced the cytotoxicity of LPS-stimulated bovine monocyte culture supernatants. Size exclusion high-performance liquid chromatography (HPLC) analysis of LPS-stimulated monocyte and alveolar macrophage culture supernatants resulted in a molecular weight elution profile similar to that of recombinant human TNF-alpha. These elution profiles are consistent with the presence of multimers of TNF-alpha. This is believed to be the first report of the in vitro production of bovine TNF-alpha.     

Candida albicans blastoconidia in peripheral blood smears from non-neutropenic surgical patients.

An 80 year old woman developed fever 11 days after volvulus surgery. A peripheral blood smear showed numerous yeast cells--both extraleucocytic and intraleucocytic--as well as leucoagglutination. The fungal elements included blastospores, pseudohyphae, and germ tubes. Two days later, blood cultures yielded Candida albicans, Enterobacter aerogenes, and Staphlococcus aureus. The patient had no medical history of immunodeficiency. Several reports indicate that fungal elements may be detected in peripheral blood smears from patients who have a severe intestinal disease.     

Mercury induces inflammatory mediator release from human mast cells

Background  

Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD) have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl₂) on human mast cell activation.     

Interleukin-1 beta release from human peripheral blood monocytes associated with phagocytosis of carbonyl-iron or erythrocytes

Interleukin-1 beta (IL-1 beta) release from human peripheral blood monocytes during the incubation with carbonyl-iron or sheep red blood cells was investigated. The incubation of purified monocytes with carbonyl-iron or sheep red blood cells enhanced IL-1 beta release, while their compounds, hemoglobin, globin and ferric citrate did not. The mechanisms of IL-1 beta release by carbonyl-iron or sheep red blood cells may be related to their phagocytosis, as non-phagocytic monocytes did not release IL-1 beta.     

Cytokine gene expression in human peripheral blood mononuclear cells stimulated by mannoprotein constituents from Candida albicans.

The expression of cytokine genes in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with mannoprotein constituents (MP) of Candida albicans has been studied by means of S1 nuclease mapping analysis, polymerase chain reaction, and enzyme-linked immunosorbent assay. MP induced early, consistent, and long-lasting production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 mRNAs. Similar results were obtained when the same PBMC cultures were stimulated with the purified protein derivative (PPD) from Mycobacterium tuberculosis or with IL-2, although lower levels of IL-6 mRNA were detected in IL-2-stimulated cells than in MP- or PPD-stimulated cells. MP, PPD, and IL-2 induced appreciable levels of granulocyte-macrophage colony-stimulating factor and gamma interferon, but only MP and PPD were able to induce IL-2 mRNA. MP were unable to stimulate a consistent expression of the genes encoding for IL-4, IL-5, and IL-10, while low, sometimes barely detectable levels of these cytokine mRNAs were observed in PPD- or IL-2-stimulated PBMC cultures. When protein synthesis of MP-stimulated PBMC was inhibited by cycloheximide, a superinduction of mRNAs for IL-4 and IL-10 and, more markedly, gamma interferon was observed. Overall, these results highlight the powerful, selective induction of cytokine gene expression by MP constituents of C. albicans in human PBMC cultures, thus providing some functional clues to explain the efficient state of the anticandidal response in normal human subjects.     

Proliferation of human peripheral blood mononuclear cells induced by Candida albicans and its cell wall fractions

Glutaraldehyde-inactivated cells and cell-wall fractions of Candida albicans were studied for their capacity to induce or inhibit the in-vitro proliferation of human peripheral blood mononuclear cells (PBMC), as measured by 3H-thymidine incorporation. Both the intact cells (CA) and a phosphorylated gluco-mannan-protein complex of the cell wall (GMP), in microgram doses, were strong inducers of PBMC proliferation, with a peak of activity at 6-9 days of culture and varying with the PBMC donor. A significant but much lower proliferation was observed on exposure of PBMC to a low-protein (less than 3% by weight) mannan component (M) of the cell wall. Both a hot-alkali extracted mannan-protein complex (M-alk), comparable to GMP in crude chemical composition, and an alkali-insoluble cell-wall glucan (GG) were inactive. None of the Candida fractions induced a lymphoproliferation of umbilical cord blood cells and all fractions, except GG, were equally effective in binding human anti-Candida antibodies as shown by a sensitive ELISA-inhibition assay. Moreover, a monoclonal antibody against the class II determinant of the HLA complex inhibited PBMC proliferation irrespective of the Candida antigen used. Taken together, the data shows that in inducing lymphoproliferation, Candida fractions act as specific antigens rather than as non-specific mitogens. Use of intact Candida cells and chemically-defined cell-wall components appears preferable to use of undefined antigenic mixtures as stimulators of PBMC proliferation.     

Hyphae and yeasts of Candida albicans differentially regulate interleukin-12 production by human blood monocytes: inhibitory role of C. albicans germination

The role of Candida albicans yeast-to-hyphae transition in interleukin-12 (IL-12) production by monocytes was investigated. Germinating C. albicans not only failed to induce IL-12 p70 but also suppressed IL-12 production induced by heat-killed C. albicans. Comparison of the abilities of germinating C. albicans and agerminating mutants to inhibit IL-12 production showed that germination of C. albicans plays a critical role in the inhibition of IL-12 production.     

Activation of human peripheral blood monocytes by lipoproteins

Activation of human peripheral blood monocytes could enhance their attachment and or migration into the arterial intima and their various secretory and other functions, thus influencing the pathogenesis of atherosclerosis. In these experiments the authors have explored the role of lipoproteins in the activation of human blood monocytes. Monocytes were purified from citrated blood by Histopaque density gradient centrifugation and countercurrent centrifugal elutriation and cultured in DMEM in the presence of 20% acid-treated autologous serum or 100 micrograms/ml each of VLDL, LDL, Ac-LDL, and HDL. Secretion of beta-glucuronidase activity into the media was measured as a marker of activation. All of the lipoprotein density classes as well as serum stimulated secretion of beta-glucuronidase activity, with LDL and Ac-LDL having a greater influence than serum, VLDL, or HDL. Serum and LDL also stimulated secretion of prostaglandin E into the culture medium. Incubation of monocytes with serum or LDL in the presence of inhibitors of arachidonate metabolism (NDGA and indomethacin) resulted in a significant decrease in secreted and intracellular beta-glucuronidase activity, indicating a role for products of arachidonate metabolism in the activation of monocytes by lipoproteins.     

Alpha,beta-unsaturated aldehydes in cigarette smoke release inflammatory mediators from human macrophages

Smoking cigarettes is the major risk factor for chronic obstructive pulmonary disease (COPD). COPD is a condition associated with chronic pulmonary inflammation, characterized by macrophage activation, neutrophil recruitment, and cell injury. Many substances contained in cigarette smoke, including reactive oxygen species (ROS), have been proposed to be responsible for the inflammatory process of COPD. However, this issue remains unsettled. By gas chromatography/mass spectrometry (GC/MS) we show that acrolein and crotonaldehyde, two alpha,beta-unsaturated aldehydes, are contained in aqueous cigarette smoke extract (CSE) at micromolar concentrations and mimic CSE in evoking the release of the neutrophil chemoattractant IL-8 and of the pleiotropic inflammatory cytokine TNF-alpha from the human macrophagic cell line U937. In addition, acrolein (10-30 microM) released IL-8 also from cultured human alveolar macrophages and THP-1 macrophagic cells. 4-hydroxy-2-nonenal (30-100 microM), an endogenous alpha,beta-unsaturated aldehyde that is abundant in lungs of patients with COPD, stimulated the release of IL-8 from U937 cells, whereas the saturated aldehyde, acetaldehyde, was ineffective. CSE-evoked IL-8 release was remarkably (> 80%) inhibited by N-acetyl-cysteine (0.1-3 mM) or glutathione monoethyl ester (1-3 mM). Both compounds, by forming covalent adducts (Michael adducts), completely removed unsaturated aldehydes from CSE. Our data demonstrate that alpha,beta-unsaturated aldehydes are major mediators of cigarette smoke-induced macrophage activation, and suggest that they might contribute to pulmonary inflammation associated with cigarette smoke.     

Borrelia burgdorferi stimulates release of interleukin-1 activity from bovine peripheral blood monocytes.

Infection with Borrelia burgdorferi is suspected to be a cause of lameness and arthritis in cattle. Interleukin-1 (IL-1) activity has been detected in joint fluids from human patients affected by various arthritides, including Lyme arthritis. In addition, human monocytes and murine macrophages have been reported to release IL-1 activity when incubated with B. burgdorferi in vitro. To address a possible mechanism by which B. burgdorferi might cause a bovine arthritic syndrome, we determined whether bovine peripheral blood monocytes released IL-1 activity when coincubated with B. burgdorferi in vitro. High-passage and low-passage isolates of B. burgdorferi stimulated release of IL-1 activity from bovine monocytes. The amount of IL-1 activity released was dependent on the number of borreliae added to the monocyte cultures. In addition, live and heat-killed B. burgdorferi cells stimulated release of similar amounts of IL-1. We also obtained no evidence that soluble components released from in vitro-cultured B. burgdorferi stimulated IL-1 release from bovine monocytes. A recombinant IL-1 receptor antagonist blocked the proliferative activity of monocyte-conditioned medium in a thymocyte costimulation assay, thus demonstrating that the costimulatory activity detected was due to IL-1.     

Time-course of the histamine release from human peripheral blood monocytes and the influence of ketotifen and disodium cromoglycate (DSCG)

Inflammation Research -     

Effect of strontium-substituted biphasic calcium phosphate on inflammatory mediators production by human monocytes

Calcium phosphate materials are widely used as bone substitutes because of their properties close to those of the mineral phase of bones. Nevertheless, after several months, calcium phosphate-based materials release particles that may be phagocytosed by monocytes, leading to an inflammatory reaction. Strontium is well known to counteract the osteoporosis process, but little is known about its effect on inflammatory processes. The purpose of this work was to study the effect of biphasic calcium phosphate (BCP) particles substituted with strontium on the inflammatory reaction. Human primary monocytes stimulated or not by lipopolysaccharide (LPS) were exposed to BCP particles containing strontium for 6 and 24 h. Inflammatory mediators (cytokines and matrix metalloproteinases (MMPs)) production was then quantified by ELISA and zymography. We observed that the presence of strontium had few effects on unstimulated cells, but it decreased the production of pro-inflammatory cytokines and the chemokine interleukin 8 in LPS-stimulated cell-conditioned medium. This work suggests for the first time that strontium may be involved in the control of inflammatory processes following BCP phagocytosis by human monocytes.     

Analysis of methods for the generation of dendritic cells from human peripheral blood monocytes

Dendritic cells (DC) are highly efficient antigen-presenting cells that initiate the primary immune response. Several laboratories have developed culture systems for human DC from peripheral blood monocytes. Most of these studies have used fetal calf serum (FCS) containing culture conditions that are inappropriate for human application. GM-CSF and IL-4 were used to make immature DC. The monocyte-conditioned medium (MCM) was used to induce the final maturation of DC. Using the previously described methods, the quality of MCM has unpredictable variations. Therefore using a defined cocktail of growth factors for the generation of mature DC would be advantageous for experimental as well as clinical purposes. In this study, it is suggested that combinations of both GM-CSF/IL-4 or GM-CSF/IL-13 could be used as the first-step culture to produce immature DC, and that cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6, PGE2) was as efficient as MCM for the second step-culture to produce fully maturated DC. Here, we have generated an easily reproducible culture system for DC that allows for the generation of large amounts of immature and mature DC, and we also now have established the method in a FCS-free system that is suitable for clinical use.     

The binding of SRBC by human peripheral blood monocytes

In a population of E-rosette forming human peripheral blood cells, about 20% of the rosettes were formed by monocytes. About 60% of the peripheral blood monocytes bound SRBC in a standard E-rosette test.