Morbidity and mortality associated with reperfusion injury post-myocardial ischemia (MI/R) occur through the action of complement. Although the Stahl laboratory has implicated mannose-binding lectin (MBL) as the initiator of complement activation in rats subjected to MI/R, their previous studies did not identify definitively the complement pathway involved. In a continuation of their work, Walsh et al. (p. 541 ) found that C57BL/6 mice lacking MBL or both C2 and factor B (fB) were protected from infarction following MI/R compared with wild-type mice; mice lacking C1q were not protected. Administration of purified MBL or C2, respectively, before ischemia abrogated the protection in the knockout animals. Left ventricular function in MBL−/− mice was retained following MI/R but was decreased in wild-type and C1q−/− controls and in MBL−/− mice given MBL. Mice lacking the alternative pathway of complement activation (fD−/−) and mice having only an active lectin pathway (lacking both classical and alternative pathways) had decreased left ventricular function compared with sham-operated animals or MI/R in mice lacking C2/fB. C1q and C3 were deposited in the infarcted tissue in wild-type and C1q−/− mice; colocalization of C3 and MBL deposits was seen in infarcted tissue in MI/R. The authors conclude that MBL-dependent lectin complement pathway activation is important in the development of MI/R injury in this mouse model.

Many autoimmune diseases, such as systemic sclerosis (SSc) and autoimmune thyroid disease (AITD), have high female/male ratios. The X chromosome carries genes that determine sex hormone levels and maintain immune tolerance. Recently, Invernizzi and collaborators suggested that sex chromosome defects might be involved in autoimmune primary biliary cirrhosis. In their study on p. 575 , the same investigators determined the frequency of monosomy X in peripheral white blood cells from 44 female patients with SSc, 45 female patients with AITD (32 with Hashimoto’s thyroiditis and 12 with Graves’ disease), and 73 healthy women of similar age. Although monosomy X increased with age at the same rate in all groups, SSc was more prevalent in older women. Monosomy X was seen at higher frequency in white blood cells of women with SSc and AITD compared with age-matched healthy controls. No differences in monosomy X rates were detected for patients with different types of SSc or AITD. Monosomy X was found at highest frequency in T and B cells, but also in monocytes/macrophages, polymorphonuclear leukocytes, and NK cells from patients with autoimmune diseases. No Y chromosome-specific sequences were detected in a subpopulation of 11 women with male offspring. The authors propose that dysregulation of genes on the X chromosome predisposes women to two autoimmune disorders, SSc and AITD, and rule out a contribution from male microchimerism.

Among the consequences of MHC interaction with the TCR:CD3 complex is phosphorylation and recruitment of signaling and adaptor molecules. Interaction of the CD3ε subunit with Nck, a cytosolic adaptor protein that interacts with over 45 different proteins, is thought to be essential for optimal T cell activation and formation of the immunological synapse. By retroviral-mediated stem cell gene transfer, Szymczak et al. (p. 270 ) developed a mouse strain with CD3ε mutated in the proline-rich sequence that binds the first of three N-terminal Src homology 3 domains in Nck. Pull-down experiments demonstrated lack of Nck:CD3ε interactions following TCR ligation of thymocytes from the mutant mice, whereas interactions were seen in thymocytes from wild-type mice. No differences between mutant and wild-type mice were detected in thymic T cell development, in numbers of peripheral CD4+ T cells, CD8+ T cells, or B cells, or in T cell functions such as CD69 expression, proliferation, or TCR internalization after TCR stimulation. The data indicate that preventing CD3ε:Nck interactions by mutating the proline-rich sequence of CD3ε has no observable impact on T cell development or function in mice.

Binding of Ag to the BCR initiates signal transduction and endocytosis of Ag. Although Vilen and collaborators have shown reduced association of BCR components, Ig- and μ-H chain (μm), following ligation, details of destabilization and fate of components and Ag are unknown. In Kim et al. (p. 147 ), the Vilen laboratory found Ig-α, but not μm, in sucrose gradient-separated lipid microdomains from Ag-stimulated mouse cells within 1 min of Ag stimulation; both components were found in anti-μm mAb immunoprecipitates from unstimulated cells or from cells treated with a Src kinase inhibitor. Colocalization of μm and Ig-β by immunofluorescence was 40% in unstimulated cells but only 14% in Ag-stimulated cells. Transmission electron microscopy on paraformaldehyde-fixed native membrane sheets from unstimulated mouse cells showed close colocalization of added gold-conjugated Ag (μm-associated) and Ig-α gold particles. Colocalization of the components was decreased 6-fold, and the distance between the components increased nearly 12-fold in cells stimulated for 5 min before fixation. By confocal and transmission electron microscopy, association of μm and Ag with clathrin-coated vesicles (CCVs) was seen to increase dramatically after stimulation and did not occur in the presence of the Src kinase inhibitor. Stimulation did not induce a change in association of Ig-β or Ig-α with CCVs. The data suggest that BCR cross-linking results in dissociation of Ig-α/β from the Ag-μm complex; the former stays within lipid microdomains, whereas the latter enters CCVs in the endocytic pathway in a Src kinase-dependent manner.

Production of anti-chromatin Abs by autoreactive B cells in lupus-prone mice is CD4+ T cell dependent. However, the impact of Th cell subsets or other factors on the differentiation pathway of activated B cells has not been delineated. Fields et al. (p. 104 ) injected mice with influenza virus and distinct subsets of hemagglutinin (HA)-specific TCR transgenic CD4+ T cells before transfer of anergic B cells, transgenic for both influenza virus HA Ag and an anti-chromatin H chain, from healthy mice. Increased numbers of anti-chromatin B cells and anti-chromatin autoantibodies were seen in mice given Th2-polarized anti-HA cells or Th1-polarized anti-HA cells producing a high level of IFN-γ (IFN-γhigh) compared with mice given B cells alone; no increases were seen in mice given Th1-polarized anti-HA cells producing a low level of IFN-γ (IFN-γlow). Anti-CD154 mAb blocked B cell recovery and anti-chromatin Ab production stimulated by either Th2 or IFN-γhigh Th1 cells. FasL-deficient Th2 or IFN-γlow Th1 cells further enhanced titers of anti-chromatin Ab, but B cell recovery was increased only in mice given FasL-deficient IFN-γlow Th1 cells. Anti-chromatin Ab-forming cells were more tightly clustered in extrafollicular foci in spleens of mice given Th2 cells vs IFN-γhigh Th1 cells; IFN-γlow Th1 cells did not induce Ab-forming cells. A higher frequency of anti-chromatin germinal centers was detected in recipients of IFN-γhigh or IFN-γlow Th1 cells compared with Th2 cells. Anti-chromatin B cells in germinal centers were increased further in recipients of FasL-deficient Th2 cells; anti-chromatin germinal centers and Ab-forming cells formed in spleens of FasL-deficient IFN-γlow Th1 cell recipients. The authors show that the ability of Th2 cells, or Th1 cells producing IFN-γ, to induce Ab production by anti-chromatin B cells is influenced by FasL.

Dendritic cells (DC) secrete factors that activate B cells and promote their differentiation into Ab-secreting cells. Macrophage secretions enhance proliferation of activated B cells. However, it is not known whether DC and macrophages also play a role in regulating B cell tolerance. Kilmon et al. (p. 37 ) discovered that purified anergic autoreactive B cells from mice expressing H and L chain transgenes produced IgM H chain in response to LPS stimulation; unpurified B cells were not responsive to LPS. Purified B cells lost responsiveness when mixed with B cell-depleted splenocytes or with purified bone marrow-derived DC (10 B cells/1 DC) or purified bone marrow-derived macrophage (100 B cells/1 macrophage). Splenic macrophages were twice as effective as splenic myeloid DC in repressing LPS-induced Ig secretion. Conditioned medium from LPS-stimulated DC or macrophage cultures inhibited Ig secretion; addition of anti-IL-6 Ab removed the inhibition only of the DC conditioned medium. rIL-6 alone suppressed Ig secretion. IL-6 or conditioned medium from LPS-stimulated DC had minimal effect on naive B cells from nontransgenic mice but suppressed Ig secretion by B cells chronically exposed to self Ags. The authors propose a model in which IL-6 produced by DC, or an unidentified factor produced by macrophages, during microbial infection activates naive B and T cells but suppresses Ig secretion by autoreactive B cells.

Chemerin, identified in a human ascitic fluid by Parmentier and colleagues, recruits APC to sites of inflammation. Neither the cell that secretes prochemerin, the inactive precursor, nor the proteolytic enzyme(s) responsible for its conversion to the active form has been identified. In further work from that laboratory, Wittamer et al. (p. 487 ) used conditioned media from human polymorphonuclear cells (PMN) degranulated with cytochalasin B and fMLP or LPS and fMLP to convert human recombinant prochemerin to chemerin, which activated its receptor in a calcium mobilization assay. Medium from unstimulated PMN had only low basal receptor activity. Serine protease inhibitors significantly decreased PMN-induced chemerin production, and the only active serine proteases of those tested were cathepsin G and human leukocyte elastase. Sites of enzyme cleavage were determined by mass spectrometry and microsequencing to be after phenylalanine 156 and serine 157, respectively. Both bioactive forms of chemerin were detected by mass spectrometry in conditioned medium from neutrophils activated by cytochalasin B and fMLP. Addition of plasma or purified secretory leukoproteinase inhibitor, a neutrophil serine protease inhibitor found in plasma, abolished receptor activation. Both chemerin variants activated human monocyte-derived immature dendritic cells. The study identifies two neutrophil-derived serine proteases responsible for processing prochemerin to active forms that attract APC to inflammatory sites.

Summaries written by Dorothy L. Buchhagen, Ph.D.