Interaction of Bartonella henselae with the murine macrophage cell line J774: infection and proinflammatory response.

Bartonella henselae is the causative agent of cat scratch disease (CSD), a self-limiting condition characterized by a subacute regional lymphadenopathy that may develop into disseminated bartonellosis in immunocompromised subjects. Mice experimentally infected with B. henselae display typical liver and spleen granulomas rich in T cells and macrophages. So far there are no data on the interaction between bartonellae and macrophages. In order to clarify this topic, we investigated the interaction of B. henselae with J774, a mouse macrophage cell line. Analysis of bacterial uptake by functional assays and transmission electron microscopy indicates that bartonellae can enter and survive inside J774. Entry occurred within 30 min postinfection and reached a plateau at 160 min. Infection of J774 was followed by a dose-dependent release of the proinflammatory cytokines tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and IL-6. Bartonellae persisted intracellularly without loss of viability for at least 8 h, and their number slightly decreased 24 h postinfection. Gamma interferon (IFN-gamma) treatment of J774 significantly decreased the number of recoverable bacteria at 8 and 24 h. This enhancement of macrophage bactericidal activity was associated with nitric oxide (NO) release and was prevented by the addition of the competitive inhibitor of NO synthesis N(G)-monomethyl L-arginine. These findings suggest that IFN-gamma-mediated activation of macrophages may be important for the clearing of B. henselae infection and that anti-B. henselae microbicidal activity of IFN-gamma-activated macrophages is mediated to a large extent by NO production.

Serum amyloid A is an activator of PMN antimicrobial functions: induction of degranulation, phagocytosis, and enhancement of anti-Candida activity.

Serum amyloid A (SAA) is a 12-kDa protein secreted in large amounts by liver cells during microbial infections or inflammatory diseases. We have recently reported that SAA induces chemotaxis of polymorphonuclear cells (PMN), monocytes, and T lymphocytes and stimulates their adhesion to endothelial monolayers. In this study, we investigated whether SAA regulates PMN antimicrobial activities. We found that recombinant SAA (rSAA), at concentrations comparable to serum levels attained during an acute phase response, is a potent activator of PMN. Stimulation of PMN by rSAA results in a rapid and transient increase of cytosolic calcium concentration and up-regulation of cell-surface expression of antigens involved in adhesion and microbial recognition such as CD11c and CD16. In addition, stimulation of PMN with rSAA increases secretion of lactoferrin, an antimicrobial protein that is contained in specific granules of PMN and enhances PMN phagocytic activity against heat-killed Candida albicans. Finally, activation of PMN with rSAA enhances their anti-Candida activity within 30 min of stimulation. These results suggest that SAA is involved in up-regulating PMN antimicrobial activities and that high circulating concentrations of SAA as seen in the acute phase response may constitute a potential host defense mechanism against fungal infections.

Expression and role of IL-15 in post-burn hypertrophic scars.

Hypertrophic scarring is a skin disorder that occurs after wounding and thermal injury. There is accumulating evidence that immunologic processes such as infiltration of activated T lymphocytes and altered cytokine production may play a role in the formation of hypertrophic scars. Interleukin-15, a cytokine identified as a T cell growth factor, also acts as a chemoattractant for T cells and has pro-inflammatory properties. We investigated the expression and the role of this cytokine in hypertrophic scarring. IL-15 expression was compared in skin biopsies of hypertrophic scars (HS) both in active (AHS) and in remission (RHS) phases, in normotrophic scars (NTS) and in normal skin using reverse transcriptase-polymerase chain reaction and immunohistochemistry. IL-15 expression in HS was significantly higher than in NTS or normal skin. Furthermore, AHS expressed higher levels of IL-15 than RHS. Immunohistologic analysis of AHS samples showed strong IL-15 immunoreactivity in keratinocytes and Langerhans cells in the epidermis and in macrophages, fibroblasts, and dermal dendritic cells in the dermis. High levels of IL-15 expression in AHS correlated with abundant infiltration of activated CD3+ cells. Ex vivo experiments indicate that IL-15 can sustain the proliferative response of T cells derived from AHS but not from RHS and NTS. In addition, IL-15 prevents both cytokine deprivation and activation-induced apoptosis of T cells derived from AHS. Taken together, these results suggest that IL-15 can be involved in the recruitment, proliferation, and apoptosis inhibition of T cells in AHS. The findings that the evolution from an AHS to a RHS is associated with a decrease in IL15 expression, and with a loss of IL-15 responsiveness in ex vivo-cultured T cells, indicate that this cytokine plays an important role in the biology of pathologic scar formation.

Human monocytes constitutively express membrane-bound, biologically active, and interferon-gamma-upregulated interleukin-15.

Interleukin-15 (IL-15) is a potent regulator of T-, B-, and natural killer cell proliferation and displays unusually tight controls of secretion. Even though IL-15 mRNA is constitutively expressed in monocytes/macrophages and is upregulated by a variety of stimuli, evidence for IL-15 cytokine secretion is only found exceptionally, eg, conditions of pathological, chronic inflammation. This raises the possibility that monocytes express membrane-bound IL-15 rather than secrete it. The current study explores this hypothesis. We demonstrate here that biologically active IL-15 is indeed detectable in a constitutively expressed, membrane-bound form on normal human monocytes, as well as on monocytic cell lines (MONO-MAC-6, THP-1, and U937), but not on human T or B cells (MT4, M9, C5966, JURKAT, DAUDI, RAJI, and Epstein-Barr virus-immortalized B-cell clones). Furthermore, cell surface-bound IL-15 is upregulated upon interferon-gamma stimulation. Interestingly, monocyte/macrophage inhibitory cytokines such as IL-4 and IL-13 fail to downregulate both constitutive and induced cell-surface expression of IL-15. Membrane-bound IL-15 does not elute with acetate buffer or trypsin treatment, suggesting that it is an integral membrane protein and that it is not associated with the IL-15 receptor complex. Finally, membrane-bound IL-15 stimulates T lymphocytes to proliferate in vitro, indicating that it is biologically active. These findings enlist IL-15 in the fairly small family of cytokines for which the presence of a biologically active membrane-bound form has been demonstrated (eg, IL-1, tumor necrosis factor-alpha, and IL-10) and invites the speculation that most of the biological effects of IL-15 under physiological conditions are exerted by the cell surface-bound form.

Interleukin-15 activates proinflammatory and antimicrobial functions in polymorphonuclear cells.

Interleukin-15 (IL-15) is a recently discovered cytokine produced by a wide range of different cell types including fibroblasts, keratinocytes, endothelial cells, and macrophages in response to lipopolysaccharide or microbial infection. This suggests that IL-15 may play a crucial role in the activation of phagocytic cells against pathogens. We studied polymorphonuclear leukocyte (PMN) activation by IL-15, evaluated as enhancement of PMN anti-Candida activity as well as IL-8 production, following stimulation with the cytokine. The PMN response to IL-15 depends on binding to the IL-15 receptor. Our experiments show that binding of a biotinylated human IL-15-immunoglobulin G2b IgG2b fusion protein was competed by the addition of human recombinant IL-15 (rIL-15) or of human rIL-2, suggesting that IL-15 binding to PMN might involve the IL-2Rbeta and IL-2Rgamma chains, which have been shown to be constitutively expressed by PMN. In addition, we show by reverse transcription-PCR and by flow cytometry with a specific anti-IL-15Ralpha chain monoclonal antibody that PMN express the IL-15Ralpha chain at the mRNA and protein levels. Incubation with IL-15 activated PMN to secrete the chemotactic factor IL-8, and the amount secreted was increased by costimulation with heat-inactivated Candida albicans. In addition, IL-15 primed the metabolic burst of PMN in response to formyl-methionyl-leucyl-phenylalanine but was not sufficient to trigger the respiratory burst or to increase the production of superoxide in PMN exposed to C. albicans. IL-15 also increased the ability of PMN to phagocytose heat-killed C. albicans organisms in a dose-dependent manner, without opsonization by antibodies or complement-derived products. In the same concentration range, IL-15 was as effective as gamma interferon (IFN-gamma) and IL-2 in increasing the C. albicans growth-inhibitory activity of PMN. Taken together, these results suggest that IL-15 is a potent stimulant of both proinflammatory and antifungal activities of PMN, activating several antimicrobial functions of PMN involved in the cellular response against C. albicans.

Interleukin-15 (IL-15) induces IL-8 and monocyte chemotactic protein 1 production in human monocytes.

Interleukin-15 (IL-15) is a recently characterized cytokine that shares many biological activities with IL-2 and interacts with the beta and gamma components of the IL-2 receptor. Unlike IL-2, which is secreted only by T cells, IL-15 is expressed preferentially by nonlymphoid tissues, epithelial, and fibroblast cell lines and by activated monocytes/macrophages. High concentrations of IL-15 have been shown in inflamed joints of rheumatoid arthritis patients, suggesting a role for IL-15 in inflammatory diseases where there is recruitment of leukocytes. Although monocytes have been shown to bind IL-15, its effects on these cells are not defined. In this report we show that supernatants of monocytes treated with IL-15-contained chemotactic activity for neutrophils and monocytes which was neutralized by anti-IL-8 or by anti-monocyte chemotactic protein 1 (MCP-1) antibodies, respectively. Secretion of IL-8 and MCP-1 proteins is detectable by enzyme-linked immunosorbent assay as early as 6 hours after stimulation with IL-15. Production of the two chemokines is correlated with induction by IL-15 of mRNA expression in monocytes. In addition, IL-8 and MCP-1 induction by IL-15 is differently regulated by interferon-gamma (IFN-gamma) and IL-4. IFN-gamma inhibited IL-15-induced IL-8 secretion, but synergized with IL-15 in MCP-1 induction; whereas IL-4 inhibited both IL-8 and MCP-1 induction by IL-15. These results show that IL-15 can stimulate monocytes to produce chemokines that cause inflammatory cell accumulation. Thus, IL-15 locally produced at sites of inflammation may play a pivotal role in the regulation of the leukocyte infiltrate.

Rubella virus hemagglutination with human and animal erythrocytes: effect of age and trypsinization.

Sensitivity to rubella virus hemagglutination differs considerably with newborn as opposed to adult erythrocytes (RBC) in several animal species. In humans, cord RBC are 16 times more sensitive to hemagglutination, whereas hemagglutination titers with newborn RBC are 6 to 16 times higher in chickens, mice, and rabbits. This higher sensitivity is, however, short-lived, and adult RBC values are observed 7 days after birth in mice. Trypsinization has no effect on newborn RBC, but increases sensitivity of adult RBC to that of neonatal RBC and tends to diminish differences among individuals. On the other hand, trypsinization does not further enhance the very high sensitivity of pigeon, duck, and goose RBC or the low sensitivity of both newborn and adult guinea pig RBC. Human cord RBC proved suitable for use in hemagglutination-inhibition tests, since they gave titers comparable to those obtained with other types of RBC and offered some advantages: by comparison with animal RBC, they did not require adsorption to remove nonspecific agglutinins; by comparison with adult human RBC, they did not need trypsinization.

[Aspecific inhibitors of rubella virus hemagglutination in normal and thyroidectomized rats]. / Inibitori aspecifici dell'emoagglutinazione da virus della rosolia in ratti normali e tiroidectomizzati

Thyroidectomized rats show alterations in serum lipoprotein distribution with an high increase of low density lipoproteins (LDL): however inhibitors titre was the same both in control and in treated animals. Also removal of nonspecific inhibitors by precipitation with heparin-MnCl2 or D.S. CaCl2 methods did not show differences between control and treated animals.