TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection.

Calcium signaling in phagocytes is essential for cellular activation, migration, and the potential resolution of infection or inflammation. The generation of reactive oxygen species (ROS) via activation of NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase activity in macrophages has been linked to altered intracellular calcium concentrations. Because of its role as an oxidative stress sensor in phagocytes, we investigated the function of the cation channel transient receptor potential melastatin 2 (TRPM2) in macrophages during oxidative stress responses induced by Helicobacter pylori infection. We show that Trpm2-/- mice, when chronically infected with H. pylori, exhibit increased gastric inflammation and decreased bacterial colonization compared with wild-type (WT) mice. The absence of TRPM2 triggers greater macrophage production of inflammatory mediators and promotes classically activated macrophage M1 polarization in response to H. pylori. TRPM2-deficient macrophages upon H. pylori stimulation are unable to control intracellular calcium levels, which results in calcium overloading. Furthermore, increased intracellular calcium in TRPM2-/- macrophages enhanced mitogen-activated protein kinase and NADPH-oxidase activities, compared with WT macrophages. Our data suggest that augmented production of ROS and inflammatory cytokines with TRPM2 deletion regulates oxidative stress in macrophages and consequently decreases H. pylori gastric colonization while increasing inflammation in the gastric mucosa.

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis.

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1(rnu/rnu) ) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.

Expression and function of CD22, a B-cell restricted molecule.

In this work, we studied the expression and function of CD22 in murine B cells. CD22 has been previously characterized as an activation marker of mature B lymphocytes. However, we found that CD22 is expressed early during the ontogeny of B cells in the bone marrow and spleen, and was found on B cells isolated from all the different lymphoid compartments. We also found that B cells stimulated through the B-cell antigen receptor (BCR), CD38 and CD40, upregulated CD22 expression to maximal levels within 24 h after stimulation, but that the levels of CD22 declined at later times (48 and 72 h). CD22 is rapidly phosphorylated after BCR signal transduction, and is believed to downregulate B-cell activation. In this study, we did not detect CD22 phosphorylation in activated B cells after CD38 or CD40 cross-linking, even though CD22 was clearly phosphorylated in the BCR-stimulated B cells. Consistent with this, we found no evidence of physical association between CD38 or CD40 and CD22 in B cells. The lack of association or phosphorylation of CD22 induced by CD38 and CD40 cross-linking indicates that CD22 may not downregulate the activation induced by these two molecules.

Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells.

Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

IgG antibody subclasses, tumor necrosis factor and IFN-gamma levels in patients with type II lepra reaction on thalidomide treatment.

A group of 9 Mexican lepromatous leprosy patients was studied at the beginning of a type II reaction (erythema nodosum leprosum, ENL) and after 1 or 2 months of thalidomide treatment. ENL patients at the onset of the reaction had slightly higher amounts of anti-Mycobacterium leprae IgG1 and IgG2 antibodies, compared to similar lepromatous patients that did not develop ENL. Neither these antibody levels nor IgM and the other IgG subclasses were importantly modified after thalidomide treatment. Serum TNF was significantly higher in the patients that developed ENL compared to those that did not develop the reaction. TNF levels were slightly decreased after 1 month of thalidomide treatment and significantly decreased after 2 months of treatment. Serum IFN-gamma was significantly lower in patients at the onset of ENL and was increased after 1 and 2 months of thalidomide treatment.

CD44-stimulated dendrite formation ('spreading') in activated B cells.

A rat monoclonal antibody (mAb) (NIM-R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes ('spreading') in B lymphocytes activated by anti-IgM and interleukin-4 (IL-4) or anti-CD38 and IL-4. In contrast, resting B cells were unable to spread similarly on the NIM-R8-coated plates. The NIM-R8 antibody recognized a 90,000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccharide, anti-IgM plus IL-4 or concanavalin A) activation. The NIM-R8 mAb with or without IL-2 or IL-4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM-R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand-inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell-surface area for cell-cell or cell-matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes.