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A subset of B lymphocytes present primarily in the peritoneal and pleural cavities is defined by the expression of CD5 and is elevated in autoimmune diseases. Upon signaling through membrane immunoglobulin M (mIgM), splenic B lymphocytes (B-2) proliferate, whereas peritoneal B cells (B-1) undergo apoptosis. However, in CD5-deficient mice, B-1 cells responded to mIgM crosslinking by developing a resistance to apoptosis and entering the cell cycle. In wild-type B-1 cells, prevention of association between CD5 and mIgM rescued their growth response to mIgM crosslinking. Thus the B cell receptor-mediated signaling is negatively regulated by CD5 in normal B-1 cells.

IntroductionJun N-terminal kinase (JNK; also known as stress-activated protein kinase, SAPK) is one of the 3 major members of the mitogenactivated protein kinase (MAPK) superfamily; the others are extracellular signal-regulated kinase (ERK) and the p38 MAP kinase. JNK is activated in response to certain growth factors or stresses such as ultraviolet (UV) radiation. Stress-induced JNK activation often leads to cell death through activation of the mitochondrial apoptotic pathway in many cell types including neuronal cells, prostate cancer cells, and fibroblasts. [1][2][3][4] On the contrary, it has been shown recently that JNK can promote survival of BCR/ABL-transformed leukemic cells. 5 Triggering the JNK pathway in vitro with a BCR-ABL tyrosine kinase led to a dramatic increase in B-cell transformation. Moreover, it was shown that JNK is required for interleukin-3 (IL-3)-mediated cell survival through its ability to phosphorylate and inactive the proapoptotic Bcl-2 family protein BAD. 6 JNK protein kinases are coded for by 3 genes, Jnk1, Jnk2, and Jnk3. Jnk1 and Jnk2 are the more widely expressed isoforms of JNK. Jnk3 is limited in expression, restricted primarily to the brain, heart, and testis. JNK is activated by upstream MAPK kinases, Activated JNK phosphorylates and activates its major substrate c-jun as well as several other transcription factors and proteins required for cell survival, proliferation, transformation, and cell death. 10 The dual role of JNK in both apoptotic and survival signaling pathways indicates that the functional role of JNK is complex. The biologic outcome of JNK activation depends upon the cellular context, time course of activation, and the balance between the ability of JNK to signal both apoptosis and cell survival. The complexity of the cellular response to JNK activation can be illustrated by the diverse actions of a proinflammatory cytokine tumor necrosis factor alpha (TNF-␣). Sustained activation of JNK correlates with TNF-induced apoptosis of rat mesangial cells. 11 On the other hand, JNK1 and JNK2 double knock-out fibroblasts are more sensitive to TNF-induced apoptosis compared with wild-type fibroblasts, suggesting a prosurvival role for JNK signaling in these cells. 12 Recent findings that MKK7 (an upstream activator of JNK) knock-out hepatocytes fail to proliferate and that mouse embryo fibroblasts that lack MKK7 undergo cellular senescence and G 2 /M growth arrest further support a role for JNK in cell-cycle progression. 13 The role of JNK during primary B-lymphocyte growth responses still awaits complete illumination. Signaling through CD72, CD40, or B-cell receptor (BCR) ligation induces activation of MAP kinases, such as JNK, in primary splenic B cells. [14][15][16] However, no defect in BCR-or CD72-induced proliferation is observed in B cells from JNK1 Ϫ/Ϫ or JNK2 Ϫ/Ϫ mice. 14 This is probably due to a redundancy of function between the 2 isoforms, as JNK1 and JNK2 double knock outs exhibit embryonic lethality. 17 In T cells, JNK2 is required for the differentiatio...

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Aged humans and rodents are susceptible to infection with Streptococcus pneumoniae bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines and increased production of interleukin (IL)-10 by macrophages (Mphi) from aged mice. To understand the molecular basis of cytokine dysregulation in aged mouse Mphi, a microarray analysis was performed on RNA from resting and lipopolysaccharide (LPS)-stimulated Mphi from aged and control mice using the Affymetrix Mouse Genome 430 2.0 gene chip. Two-way ANOVA analysis demonstrated that at an overall P < 0.01 level, 853 genes were regulated by LPS (169 in only the young, 184 in only the aged, and 500 in both). Expression analysis of systematic explorer revealed that immune response (proinflammatory chemokines, cytokines, and their receptors) and signal transduction genes were specifically reduced in aged mouse Mphi. Accordingly, expression of Il1 and Il6 was reduced, and Il10 was increased, confirming our previous results. There was also decreased expression of interferon-gamma. Genes in the Toll-like receptor-signaling pathway leading to nuclear factor-kappaB activation were also down-regulated but IL-1 receptor-associated kinase 3, a negative regulator of this pathway, was increased in aged mice. An increase in expression of the gene for p38 mitogen-activated protein kinase (MAPK) was observed with a corresponding increase in protein expression and enzyme activity confirmed by Western blotting. Low doses of a p38 MAPK inhibitor (SB203580) enhanced proinflammatory cytokine production by Mphi and reduced IL-10 levels, indicating that increased p38 MAPK activity has a role in cytokine dysregulation in the aged mouse Mphi.

The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine–inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc–inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.

A reduction in macrophage (MPhi) function with aging makes mice less responsive to bacterial capsular polysaccharides, such as those present in the pneumococcal polysaccharide vaccine, a model of thymus independent (TI) antigen (Ag). Using trinitrophenol (TNP)-lipopolysaccharide (LPS) and TNP-Ficoll, two other well-studied TI Ag, we studied the mechanistic basis of reduced MPhi function in the aged. We show that aged mice are profoundly hyporesponsive to these TI Ag. As a result of a requirement for MPhi, highly purified B cells from young-adult mice do not respond to TI Ag. When purified, young B cells were immunized with TNP-Ficoll, the antibody production from those cultures reconstituted with MPhi from aged mice was significantly lower than that seen with young MPhi. Consequently, this unresponsiveness can be overcome by a mixture of interleukin (IL)-1beta and IL-6. Upon stimulation with LPS, in comparison with young MPhi, aged MPhi secreted reduced amounts of IL-6, tumor necrosis factor alpha, IL-1beta, and IL-12, cytokines necessary for B cells to respond to TI Ag. LPS also induced aged MPhi to produce an excess of IL-10. Neutralization of IL-10 enhanced the production of proinflamatory cytokines by MPhi upon LPS stimulation and also induced Ab production by aged splenocytes. Thus, the inability of aged MPhi to help the B cell response appears to be caused by an excess of IL-10. As aged MPhi have a reduced number of cells expressing Toll-like receptor 4 and CD14, the imbalance in cytokine production might be partly a result of fewer cells expressing key components of the LPS receptor complex.

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