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Salmonella infection is associated with the increased expression of inducible nitric oxide synthase in macrophages and other cells. This review summarizes current knowledge of the molecular mechanisms involved in the induction process, and discusses the functional significance of nitric oxide production in the context of host defense against Salmonella.

When Salmonella enterica invades mammalian cells, it activates signals leading to increased expression of inflammatory mediators. One such mediator is nitric oxide (NO), which is produced under control of the enzyme inducible NO synthase (iNOS). Induction of iNOS in response to Salmonella infection has been demonstrated, but the bacterial effector molecules that regulate expression of the enzyme have not been identified. In the study reported here, an analysis of Salmonella-dependent iNOS expression in macrophages was carried out. Wild-type Salmonella strains increased the levels of both iNOS protein and mRNA in murine macrophage cell lines in an invasion-independent fashion. Mutant strains lacking a functional pathogenicity island 1-encoded type III secretion system, as well as strains lacking the invasins SipB, SipC, and SipD, were impaired in iNOS induction. Complementation experiments indicated that all three of the invasins were required for induction of iNOS expression. These results suggested that an effector protein, translocated into macrophages via the type III secretion system in a SipB-, SipC-, and SipD-dependent manner, might be the ultimate mediator of iNOS induction. In keeping with this idea, a mutant strain deficient in SopE2, a recently described homolog of SopE, was found to be impaired in the induction of iNOS expression. These observations suggest that iNOS expression is regulated by signals activated by SopE2 (and possibly SopE) and that the role of SipB, SipC, and SipD in this process is to facilitate translocation of the relevant effector.

TRAF2 is a signal transducing adaptor molecule which binds to the CD40 cytoplasmic domain. We have found that it is phosphorylated, predominantly on serine residues, when transiently overexpressed in 293 cells. The phosphorylation appears to be related to the signaling events that are activated by TRAF2 under these circumstances, since two nonfunctional mutants were found to be phosphorylated significantly less than the wild-type protein. Furthermore, the phosphorylation status of TRAF2 had significant effects on the ability of the protein to bind to CD40, as evidenced by our observations that the CD40 cytoplasmic domain interacted preferentially with underphosphorylated TRAF2 and that phosphatase treatment significantly enhanced the binding of TRAF2 to CD40. We conclude from these studies that the phosphorylation of TRAF2 is likely to play an important role in regulating signaling by virtue of its ability to influence the CD40-TRAF2 interaction., (Copyright 1999 Academic Press.)

TNF receptor-associated factor 2 (TRAF2) is a signal-transducing protein associated with the CD40 cytoplasmic domain. It has been hypothesized that during signal transduction, TRAF2 must be released from CD40 in order for it to interact with downstream signaling molecules. We found that CD40 and TRAF2 were constitutively associated with each other in a human B cell line. Following stimulation with an anti-CD40 Ab, a decrease in the amount of CD40-associated TRAF2 was observed that could not be explained by a change in total level of either of the proteins. These results, as well as similar findings obtained with 293 cells overexpressing CD40 and TRAF2, suggested that CD40-mediated signals inhibited the CD40-TRAF2 interaction. We then conducted binding studies using CD40 cytoplasmic domain fusion proteins and TRAF2 derived from either control or CD40-stimulated cell lines. These in vitro studies also indicated that the binding of TRAF2 to the CD40 cytoplasmic domain was inhibited by CD40 stimulation. The results of these experiments, as well as differences between the in vitro and in vivo findings, indicated that multiple mechanisms were involved in the inhibition of the CD40-TRAF2 interaction by CD40 signals. Possible mechanisms of inhibition are discussed based on mapping of the TRAF2 binding site on the CD40 cytoplasmic domain.

Proteins that bind to discrete domains of the Blk, Fyn, Lyn, and Btk protein tyrosine kinases were examined in pre-B cells that had not been subjected to any external stimulation, as well as in nonstimulated and antigen-receptor-ligated B cells. Proteins that bind to the Src homology 2 domains of Blk and Fyn were identified in B cells that had been activated with anti-IgM but were not identified in unstimulated B cells. A number of Blk and Fyn Src homology 2 domain-binding phosphoproteins were also observed in pre-B cells that had not been stimulated in vitro. The phosphoproteins seen in activated B cells potentially represent substrates that play a role in the pathway of antigen-receptor-mediated signaling. Distinct signaling pathways involving distinguishable kinase substrates may be relevant in pre-B-cell-receptor-mediated cell survival during ontogeny. These results indirectly support models that predict constitutive ligand-independent signaling by the pre-antigen receptor during lymphoid ontogeny.

The micron IgH chain is one component of the membrane IgM complex, the endocytic and signal transducing receptor for Ag on the surface of B lymphocytes. It is transported to the cell surface in association with three other B cell-specific proteins, an L chain and the products of the mb-1 and B29 genes. The roles played by these various proteins in mediating the functions of the complex are unclear. To analyze micron function in the absence of other lymphoid-specific proteins, we first attempted to express micron on the surface of nonlymphoid cells. Deletion of the CH1 domain was sufficient to allow surface expression of this protein in transfected COS cells as well as in mouse L cells. To determine whether this extracellularly truncated micron was capable of endocytosis, we used an assay to detect the internalization of anti-mu antibody bound to the surface of transfected cells expressing the protein. Under both cross-linking and non cross-linking conditions, the CH1-deleted micron protein was internalized in endocytic vesicles. We conclude from these observations that a) the CH1 domain of micron contains a retention signal, the elimination of which allows surface transport of this protein, and b) micron by itself is capable of at least one of the functions of the membrane IgM complex.

Mac-2, a galactose-binding lectin secretion by activated macrophages, is the major non-integrin laminin-binding protein in these cells. Mac-2 is also expressed by epithelial cells in the intestine and kidney. We wished to identify intestinal glycoproteins other than laminin that have a high affinity for Mac-2 and that could be considered as candidate ligands or partners for this lectin in intestinal epithelium. Certain lines of human colon adenocarcinoma cells produce two Mac-2-binding glycoproteins (M2BP-1 and M2BP-2) that were identified by their avid association with Mac-2 following detergent lysis and immunoprecipitation. These glycoproteins do not share a common epitope with Mac-2, and the interaction between Mac-2 and these proteins is mediated through the carbohydrate-binding domain of Mac-2 and sugar moieties on M2BP-1 and M2BP-2. M2BP-1 (98 kDa) and M2BP-2 (70 kDa) were purified by immunoaffinity chromatography and were specifically eluted with either galactose or lactose. Peptide maps revealed that M2BP-1 and M2BP-2 are structurally related. M2BP-1 is secreted and could conceivably associate with Mac-2 extracellularly. N-terminal sequence analysis of M2BP-2 suggests that these glycoproteins represent a unique subset of candidate ligands for this mammalian beta-galactoside lectin.

The membrane immunoglobulin heavy chain (micron) plays a feedback role during the pre-B stage of B lymphocyte differentiation. In pre-B cell lines, micron associates with two surrogate light chain proteins. The omega chain is disulfide linked to mu and was predicted to be the product of the lambda 5 gene. The iota chain is noncovalently associated with micron. We demonstrate that the omega protein is indeed the product of the lambda 5 gene and that mu, omega, and iota are coassociated in the same complex. Antibodies against the omega/lambda 5 protein demonstrate the existence of a subpopulation of "transitional" bone marrow B cells that express micron and omega on the cell surface. The majority of these cells also express surface kappa light chains, indicating that in B lymphoid ontogeny the lambda 5 gene is inactivated after the onset of kappa light chain expression.

The murine Mac-2 protein is a galactose- and IgE-binding lectin secreted by inflammatory macrophages. We describe here the cloning and characterization of a cDNA representing the human homolog of Mac-2 (hMac-2). The amino acid sequence derived from the hMac-2 cDNA indicates that the protein is evolutionarily highly conserved, with 85% of its amino acid residues being similar to those in the murine homolog. This conservation is especially marked in the carboxyl-terminal lectin domain. The amino-terminal half of the protein is less conserved but still contains the repetitive proline-glycine-rich motif seen in the mouse protein. hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 and binds to the desialylated glycoprotein asialofetuin and to laminin, a major component of basement membranes. These findings are discussed in the context of the potential functions of hMac-2.

The gene for a 28-kDa Mycobacterium leprae protein Ag, a major target of antibodies from patients with lepromatous leprosy, was cloned from a lambda-gt11-M. leprae DNA expression library and sequenced. Antibodies to this protein were detected in the serum of the majority of 15 individual lepromatous patients that were tested. The predicted amino acid sequence of the 28-kDa protein suggests that it is localized to the bacterial plasma membrane or cell wall.

A cDNA encoding the Mac-2 antigen, a surface marker highly expressed by thioglycollate-elicited macrophages, has been cloned by immunoscreening of a lambda gt11-P388D1 expression library. The nucleotide sequence of the cDNA is identical to that of carbohydrate-binding protein 35, a galactose-specific lectin found in fibroblasts and highly homologous to a rat IgE-binding protein from basophilic leukemia cells. The in vitro synthesized Mac-2 protein displayed the expected carbohydrate- and IgE-binding properties. By pulse-chase analysis and subcellular fractionation studies, the Mac-2 protein was found in the cytosol but was also seen to accumulate in the extracellular medium. The latter finding was surprising in view of the fact that the cDNA did not encode a signal peptide or transmembrane domain. An alternatively spliced cDNA with the potential to encode a NH2 terminally extended Mac-2 protein with a stretch of hydrophobic amino acids at its NH2 terminus was also found, but it is not clear whether it is the source of the extracellular Mac-2. Possible functions for the Mac-2 protein based on its lectin- and IgE-binding properties are discussed.