bluuebunny/crossref_metadata_2025 huggingface dataset:
Functions and specificity of T cells following nucleic acid vaccination of mice against Mycobacterium tuberculosis infection.
Authors: Zhu X , Venkataprasad N , Thangaraj H S , Hill M , Singh M , Ivanyi J , Vordermeier H M
Published: December 2022

The 38-kDa glycolipoprotein of Mycobacterium tuberculosis has been known to evoke prominent T cell and Ab responses in patients with active tuberculosis. In this study, we investigated its protective capacity using plasmid DNA immunization in a mouse experimental model. Prior knowledge of several antigenic determinants has been beneficial for analyzing the phenotype and specificity of T cells, which determine the efficacy of this vaccination procedure. C57BL/6 mice responded to the 38-kDa gene-pcDNA3 plasmid with strong CD4+ Th1 and CD8+ cytotoxic T cell responses of the IFN-gamma-producing Tc1 phenotype. After challenge with virulent tubercle bacilli, the bacterial load in the spleens and lungs of vaccinated mice was reduced to a level similar to that imparted by Mycobacterium bovis Bacille Calmette-Guérin vaccination. Notably, the specificity of CD4+ and CD8+ T cells from DNA-vaccinated and tubercle-infected mice was found to be strikingly different in respect of several peptide epitopes. The identified peptides recognized by T cells from protected mice are of further interest for the development of subunit-based vaccines against tuberculosis.
Impaired activation of nuclear factor-kappaB in endotoxin-tolerant rats is associated with down-regulation of chemokine gene expression and inhibition of neutrophilic lung inflammation.
Authors: Blackwell T S , Blackwell T R , Christman J W
Published: December 2022

We postulated that repeated injections of endotoxin could induce a state of endotoxin tolerance in rats that is mediated at least partially through a nuclear factor (NF)-kappaB-dependent mechanism. We treated rats with four doses of endotoxin (0.06 or 0.6 mg/kg/day) and evaluated the ability of these treatments to modulate activation of the transcription factor NF-kappaB induced by treatment with high dose endotoxin (6 mg/kg). In lung tissue, NF-kappaB activation in response to i.p. injection of high dose endotoxin was blocked by treatment with four daily doses of endotoxin at 0.6 mg/kg/day. Endotoxin tolerance in rats was associated with diminished endotoxin-induced mRNA expression of the NF-kappaB-dependent rat chemokine, cytokine-induced neutrophil chemoattractant. Treatment of rats with four daily doses of 0.6 mg/kg/day of endotoxin almost completely abolished the neutrophilic alveolitis that occurs in rats following i.p. injection of high dose endotoxin. These data indicate that in vivo tolerance to endotoxin challenge is associated with and possibly mediated by inhibition of NF-kappaB activation. Potentially, endotoxin tolerance could be exploited to limit inflammation in disease states whose pathobiology is related to excessive or unregulated inflammation.
Extracellular release of the type I intracellular IL-1 receptor antagonist from human airway epithelial cells: differential effects of IL-4, IL-13, IFN-gamma, and corticosteroids.
Authors: Levine S J , Wu T , Shelhamer J H
Published: December 2022

Three IL-1R antagonists (IL-1Ra) exist: secreted IL-1Ra and intracellular IL-1Ra (icIL-1Ra) types I and II. We have previously reported that human airway epithelial cells (HAEC) express icIL-1Ra type I, which can be up-regulated by corticosteroids. This study assessed whether cytokines and corticosteroids differentially effect icIL-1Ra type I protein release from HAEC to the extracellular compartment. We report that icIL-1Ra type I mRNA and intracellular protein are up-regulated in NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, in response to IL-4, IL-13, IFN-gamma, and dexamethasone. The icIL-1Ra type I protein was detected in concentrated cell culture supernatants from NCI-H292 cells and normal human bronchial epithelial cells. The release of biologically relevant concentrations of active IL-1Ra from normal human bronchial epithelial cells was demonstrated by the ability of a neutralizing anti-IL-1Ra Ab to augment IL-1beta-mediated IL-8 secretion. IL-4, IL-13, and IFN-gamma induced immunoreactive IL-1Ra release into supernatants from NCI-H292 cells. Dexamethasone inhibited constitutive and cytokine-induced release of immunoreactive IL-1Ra. The release of icIL-1Ra type I protein was not related to cytotoxicity, as measured by lactate dehydrogenase. We propose that icIL-1Ra type I release from HAEC represents a novel mechanism by which IL-1 bioactivity in the airway microenvironment may be modulated. Cytokine-mediated icIL-1Ra type I synthesis may increase both intracellular protein and release to the extracellular space, where cell surface IL-1R can be antagonized. In contrast, corticosteroid-induced increases in icIL-1Ra type I synthesis and inhibition of extracellular protein release promote accumulation of icIL-1Ra type I protein within the intracellular compartment.
Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions.
Authors: Tridandapani S , Chacko G W , Van Brocklyn J R , Coggeshall K M
Published: December 2022

To elucidate the molecular basis for inhibition of B cell proliferation and differentiation by the Fc receptor for IgG (Fc(gamma)RII), we compared the signaling events in B cells stimulated by cross-linking surface Ig alone (positive signaling), or by co-cross-linking surface Ig and Fc(gamma)RII (negative signaling). Both modes of stimulation induced tyrosine kinase activation. Positive signaling induced activation of Ras, Raf-1 kinase, and mitogen-activated protein kinase; these events were significantly attenuated during negative signaling. Since Ras is activated by SOS and Vav, two known guanine nucleotide exchange factors, activation events associated with these molecules using the two different stimuli were examined. Results of these experiments indicated that tyrosine phosphorylation of Vav did not change upon co-cross-linking. In contrast, the association of Shc and Grb2 was abrogated under negative and induced under positive signaling conditions. Concomitantly, Shc was observed to associate with a tyrosine-phosphorylated 145-kDa protein, previously identified as Src homology 2-containing inositol phosphatase, only under conditions of negative signaling. Based on these results, we hypothesize that negative signaling via the Fc(gamma)RII in B cells is at least partly the result of a block in Ras activation, and that SOS, but not Vav, is the major guanine nucleotide exchange factor in B cells for Ras activation.
Down-regulation of terminal deoxynucleotidyl transferase by Ig heavy chain in B lineage cells.
Authors: Wasserman R , Li Y S , Hardy R R
Published: December 2022

The enzyme terminal deoxynucleotidyl transferase (TdT) adds nontemplate-derived nucleotides (N regions) to the junctions between recombining variable, diversity, and joining segments of Ig genes. The relative paucity of N regions in Ig light chains, together with the down-regulation of TdT transcription in pre-B cells (prior to light chain production), suggested that production of IgM heavy chain (mu) protein might negatively regulate TdT expression. In this study, we examined the effect of mu production on TdT gene expression in B lineage subsets from normal mice, from recombination-deficient mice (SCID and Rag-1-) carrying mu transgenes, and in transformed pro-B cell lines transfected with mu constructs. In normal mice, TdT is sharply down-regulated at the early pre-B stage in which cells have just completed productive mu rearrangement. Furthermore, the expression of mu transgenes in pro-B stage cells from recombination-deficient mice results in a similar decrease. Finally, transfection of genomic constructs encoding mu into pro-B cell lines results in a marked reduction of TdT expression. Taken together, these findings indicate that mu protein production results in the down-regulation of TdT. The ability of mu transgenes to alter TdT expression in cell lines also suggests that signaling through the pre-B receptor does not necessarily require interaction with an external stromal cell-derived ligand.
The Bop gene adjacent to the mouse CD8b gene encodes distinct zinc-finger proteins expressed in CTLs and in muscle.
Authors: Hwang I , Gottlieb P D
Published: December 2022

The Bop gene (for CD8b opposite) is located immediately upstream of the mouse CD8b gene. Expression of Bop gene transcripts was previously observed in several long term CTL lines and in thymus. The present studies demonstrate that expression of the Bop gene in lymphocytes appears to be confined to CD8-positive cells, and that Bop gene expression is inducible by Con A. They further show that a single Bop gene encodes protein products with distinct amino-terminal sequences that are expressed in CTLs (t-BOP) and in cardiac and skeletal muscle (skm-BOP), as well as what appears to be a noncoding cDNA (t-ncb) expressed only in CTLs. The t-BOP and t-ncb cDNAs in CTLs result from alternative splicing of a single primary transcript, whereas the Bop transcripts expressed in CTLs and in muscle appear to be transcribed from different promoters. The BOP proteins expressed in CTLs and muscle contain zinc finger-like motifs with homology to those of the ETO/MTG8 proto-oncogene and several other proteins of interest. Western blot analysis with a hamster anti-BOP mAb have detected the BOP protein in muscle cells and in COS 7 cells transfected with Bop cDNA constructs.
Characterization of the TCRB chain repertoire in the New World monkey Callithrix jacchus.
Authors: Uccelli A , Oksenberg J R , Jeong M C , Genain C P , Rombos T , Jaeger E E , Giunti D , Lanchbury J S , Hauser S L
Published: December 2022

Callithrix jacchus is an outbred New World primate characterized by a naturally occurring bone marrow chimerism, restricted polymorphism at many MHC loci, and unusual susceptibility to viral pathogens, adenocarcinoma, colitis, and, following immunization with myelin antigens, a demyelinating disease of the central nervous system closely resembling human multiple sclerosis. Here we characterize the TCRB repertoire in this species, representing the first such analysis in a New World monkey. Two TCRBC, 13 BJ, 2 BD, and 15 BV genes were identified. Overall, a high degree of similarity with human TCRBV-D-J-C gene sequences was observed, indicating a close phylogenetic relationship. Biased usage in favor of genes from the TCRBC1-BJ1 cluster was present in 77% of sequences, in contrast to preferential usage of BC2-BJ2 genes known to occur in humans and mice. Complementarity-determining region 3 averaged 10 amino acids in length and were diverse. Framework regions of TCRBV genes were extensively conserved. Phylogenetic analysis of TCRBV sequences from different species indicated that TCR genes are highly stable across primates. Thus, a diverse TCRB repertoire is generated in C. jacchus despite the limited polymorphism of class I MHC loci. Extensive homology to human TCR genes, natural chimerism, and susceptibility to inflammatory disorders are characteristics of C. jacchus that create a useful model system for the study of human autoimmunity.
On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis.
Authors: Thomas J , Gangappa S , Kanangat S , Rouse B T
Published: December 2022

Corneal infection with herpes simplex virus-1 in immunocompetent mice induces an immunopathologic response termed herpetic stromal keratitis (HSK). The earliest sign of disease is neutrophil infiltration, which lasts for 48 to 72 h and then disappears. However, a secondary neutrophil infiltration, this time more massive, occurs, beginning 8 to 9 days postinfection, a time in which HSK becomes clinically evident. The role of neutrophils in HSK expression was investigated by eliminating such cells using a specific mAb (RB6-8C5). In neutrophil-depleted immunocompetent mice, virus replicated more abundantly, but no effects on HSK expression were observed, possibly because sustained neutropenia could not be maintained. However, using a severe combined immunodeficient mouse model, in which HSK does not occur unless given adoptive transfer of CD4+ T cells, the effects of neutrophil depletion were more pronounced. There were significantly less incidence and severity of HSK in CD4+ T cell-reconstituted severe combined immunodeficient mice that were depleted of neutrophils as compared with controls. Neutrophil-depleted mice displayed moderate to severe periocular skin lesions, progressively became cachetic, and developed signs of encephalitis. Virus was recovered at higher titers and for longer periods from eyes of neutrophil-depleted animals. Brain virus titers were also significantly higher on day 12 postinfection as compared with control animals. These results suggest that herpes simplex virus infection of the cornea rapidly invokes recruitment of neutrophils that may aid in viral clearance, and that neutrophils directly or indirectly serve as agonists in perpetuating a CD4+ T cell-mediated inflammatory reaction.
Down-regulation of Fc(epsilon)RI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody.
Authors: MacGlashan D W , Bochner B S , Adelman D C , Jardieu P M , Togias A , McKenzie-White J , Sterbinsky S A , Hamilton R G , Lichtenstein L M
Published: December 2022

Treatment of allergic disease by decreasing circulating IgE with anti-IgE Abs is currently under clinical study. Based on previous unrelated studies, it appeared likely that Fc(epsilon)RI expression on basophils and mast cells might also be regulated by levels of circulating IgE Ab. Therefore, the expression of IgE and Fc(epsilon)RI on human basophils was examined in 15 subjects receiving humanized anti-IgE mAb intravenously. Treatment with the anti-IgE mAb decreased free IgE levels to 1% of pretreatment levels and also resulted in a marked down-regulation of Fc(epsilon)RI on basophils. Median pretreatment densities of Fc(epsilon)RI were approximately 220,000 receptors per basophil and after 3 mo of treatment, the densities had decreased to a median of 8,300 receptors per basophil. Flow cytometric studies, conducted in parallel, showed similar results and also showed in a subset of 3 donors that receptors decreased with a t1/2 of approximately 3 days. The responsiveness of the cells to IgE-mediated stimulation using anti-IgE Ab was marginally decreased (approximately 40%) while the response of the same cells to stimulation with dust mite Ag, Dermatophagoides farinae, was reduced by approximately 90%. One possible explanation for these results is that Fc(epsilon)RI density is directly or indirectly regulated by plasma-free IgE levels.
IL-2 signaling controls actin organization through Rho-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-zeta.
Authors: Gómez J , García A , R-Borlado L , Bonay P , Martínez-A C , Silva A , Fresno M , Carrera A C , Eicher-Streiber C , Rebollo A
Published: December 2022

IL-2 and IL-4 induce proliferation of TS1 alpha beta cells. Activation of the zeta isoform of protein kinase C is an important step in IL-2-, but not IL-4-mediated proliferation. In addition, protein kinase C-zeta is implicated in IL-2-mediated actin organization. Given the established involvement of the Rho family of small guanine nucleotide-binding proteins in organization of actin structures, we analyze the possible relationships between Rho and protein kinase C-zeta. Using the Rho-like protein family-specific toxin B from Clostridium difficile, we report in this work that IL-2, but not IL-4, induces a Rho-dependent activation of protein kinase C-zeta. This signaling event is mediated by the activation of phosphatidylinositol 3-kinase. In contrast, IL-4 induces a Rho-independent, phosphatidylinositol 3-kinase-mediated activation of protein kinase C-zeta, but this pathway has no implications in cytoskeleton organization.
Lymphotactin is produced by NK cells and attracts both NK cells and T cells in vivo.
Authors: Hedrick J A , Saylor V , Figueroa D , Mizoue L , Xu Y , Menon S , Abrams J , Handel T , Zlotnik A
Published: December 2022

Injection of lymphotactin (Lptn) into the peritoneum caused an influx of lymphocytes at 24 h. Phenotypic analysis of the cellular influx showed that a large proportion of these cells were T lymphocytes; however, a large number of NK cells was also present. This effect of murine Lptn (mLptn) was specific since the cellular influx was blocked with a mLptn-specific mAb. Similar results were observed when Lptn was injected s.c. and the tissue was analyzed by immunohistochemistry using an anti-CD3epsilon mAb. Microchemotaxis assays confirmed that murine NK cells respond to mLptn, and also showed human NK clones to be similarly responsive to recombinant human Lptn (rhLptn). Immunohistochemical analysis of IL-2-activated murine NK cells and Northern analysis of human NK clones revealed that these cells also produce Lptn, suggesting that a self-regulatory migration mechanism exists in NK cells. Together these data confirm, in vivo, the lymphocyte specificity of Lptn previously observed in vitro and extend its chemotactic effects to the NK cell lineage. We also investigated the functional consequences of truncating the carboxyl terminus of hLptn. This truncated molecule (which is missing the carboxyl-terminal 22 amino acids of hLptn) had no detectable activity on human PBLs. In addition, while hLptn was found to attract murine splenocytes in vitro, the carboxyl-terminal truncated hLptn was again inactive on murine splenocytes. This observation indicates the presence of structural features in the carboxyl terminus of Lptn that are necessary for its biologic activity.
Integrin-mediated intracellular Ca2+ signaling in Jurkat T lymphocytes.
Authors: Weismann M , Guse A H , Sorokin L , Bröker B , Frieser M , Hallmann R , Mayr G W
Published: December 2022

T lymphocytes interact with components of the extracellular matrix after transendothelial migration on their way to sites of inflammation. To characterize the molecular basis of the interaction between T lymphocytes with different extracellular matrix proteins, we investigated the role of intracellular Ca2+ as a signal mediating such interactions and identified the cell surface integrins involved in this process. When Jurkat T lymphocytes loaded with the calcium-sensitive fluorescent dye fura-2 were placed on coverslips coated with human fibronectin, human collagen types I, IV, and VI, human tenascin, human laminin I, or mouse laminin I, an elevation in intracellular Ca2+ concentration was observed. In contrast, contact of the Jurkat T lymphocytes with vitronectin and thrombospondin did not induce Ca2+ signals in more cells as compared with control measurements in which cells were in contact with only BSA or polylysine. Furthermore, the percentage of Jurkat T lymphocytes responding with Ca2+ signals to collagen types I and IV, fibronectin, and laminin I was completely reduced to levels observed on BSA or polylysine when the cells were pretreated with specific anti-integrin Abs, suggesting a role for cell surface integrins as mediators of cell matrix-induced intracellular Ca2+ signaling. Similar results were obtained with peripheral human T lymphocytes activated by phytohemagglutinin.
Cytokine-induced VCAM-1 and ICAM-1 expression in different organs of the mouse.
Authors: Henninger D D , Panés J , Eppihimer M , Russell J , Gerritsen M , Anderson D C , Granger D N
Published: December 2022

The dual radiolabeled mAb technique was used to quantify the constitutive and induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the microvasculature of different organs of the mouse. The constitutive expression of both adhesion molecules varied significantly between tissues, with ICAM-1 levels consistently higher than VCAM-1 in all tissues studied. Following systemic administration of endotoxin (LPS), an increased surface expression of both adhesion molecules occurred in most organs, with the largest increases for ICAM-1 (2 to 3x increase) noted in the heart, small intestine, and brain, while heart and small intestine exhibited the largest increases in LPS-induced VCAM-1 expression (2 to 5x increase). These responses occurred in the face of an unaltered expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) in all tissues. TNF-alpha also elicited an increased expression of both adhesion molecules, with initial increases noted at 2 to 5 h, peak levels at 5 to 9 h, and a sustained elevation above baseline at 24 h. The TNF-alpha-induced increases in both ICAM-1 and VCAM-1 were dose dependent, with significant up-regulation noted at 5 microg/kg and maximal increases occurring at 10 to 25 microg/kg. These studies indicate that while there are significant quantitative differences in constitutive and induced expression of murine ICAM-1 and VCAM-1, the kinetics and dose-response characteristics of the two adhesion molecules to TNF-alpha are qualitatively similar.
Characteristic T helper 2 T cell cytokine abnormalities in autoimmune lymphoproliferative syndrome, a syndrome marked by defective apoptosis and humoral autoimmunity.
Authors: Fuss I J , Strober W , Dale J K , Fritz S , Pearlstein G R , Puck J M , Lenardo M J , Straus S E
Published: December 2022

Autoimmune lymphoproliferative syndrome (ALPS) is marked by massive lymphadenopathy, hepatosplenomegaly, autoimmunity and the presence of increased numbers of circulating and tissue TCR-alpha beta, CD4- CD8- T cells. The underlying defect is that of decreased T cell and B cell apoptosis, due in most, but not all, cases to heterozygous mutations of the Fas gene and corresponding defective Fas signaling function. Here we measure in vivo and in vitro cytokine secretion in ALPS to shed light on the relation of apoptosis defects to the development of autoimmunity. In in vivo studies, ALPS patients manifested greatly increased circulating levels of IL-10 (> 100-fold), compared with both healthy individuals and various disease controls; in contrast, their levels of IL-1 beta, IL-4, and IFN-gamma were normal and their levels of IL-2 and TNF-alpha were marginally increased. In parallel in vitro studies, ALPS patients CD4+ DR+ T cells stimulated either with anti-CD3/CD28 or anti-CD2/CD28 produced increased amounts of IL-4 and IL-5 (10 to 20-fold) and decreased amounts of IFN-gamma (4-fold) as compared with those of control CD4+ DR+ T cells. In contrast, ALPS patients' CD4-/CD8- T cells produced very low amounts of cytokines. Finally, ALPS patients' peripheral monocytes/macrophages produced decreased amounts of IL-12 (30-fold) and increased amounts of IL-10 (5-fold). In conclusion, ALPS is marked by the presence of DR+ T cells that exhibit a skewed Th2 cytokine response upon various forms of stimulation. This cytokine response, in the presence of increased circulating IL-10 levels, is likely to define the cytokine milieu that accounts for the humoral autoimmune features of ALPS and, perhaps, of other humoral autoimmune states.
Lipoprotein from Yersinia enterocolitica contains epitopes that cross-react with the human thyrotropin receptor.
Authors: Zhang H , Kaur I , Niesel D W , Seetharamaiah G S , Peterson J W , Prabhakar B S , Klimpel G R
Published: December 2022

Yersinia enterocolitica has recently been shown to produce a low molecular mass envelope protein that contains an epitope(s) that is cross-reactive with the extracellular domain of the human thyrotropin receptor (ETSHR). In this study, we have generated mAb to this cross-reactive protein and have obtained amino acid sequences for peptide fragments obtained from Lys-c digestion of the protein. The amino acid sequences of these peptides were identical to sequences present in bacterial lipoprotein (LP). All bacteria of the Enterobacteriaceae family produce LP as a major outer membrane protein. However, the ETSHR cross-reactive epitope(s) was shown to be unique to LP produced by Yersinia species. This was shown by Western blot analysis using a mAb specific for LP and with affinity-purified Ab specific for either LP or ETSHR and obtained from mouse antiserum generated to Y. enterocolitica. LPs from different Gram-negative bacteria were shown to be mitogenic for C3H/HeJ spleen cells and induced production and secretion of significant levels of Ig. Production of Ab that recognized the ETSHR was only induced in spleen cells stimulated with the LP obtained from Yersinia. In contrast, LP was not mitogenic for either human PBMC or human B cells. However, LP did induce IL6 and IL8 production in human monocytes at levels equivalent to that seen after LPS activation. These results identify, for the first time, the Yersinia envelope protein that is cross-reactive with the ETSHR and show that it can activate human monocytes. These findings are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the thyrotropin receptor.
Reciprocal regulation of CD30 expression on CD4+ T cells by IL-4 and IFN-gamma.
Authors: Nakamura T , Lee R K , Nam S Y , Al-Ramadi B K , Koni P A , Bottomly K , Podack E R , Flavell R A
Published: December 2022

Prior studies have implicated CD30 as a marker for Th2 cells, but the mechanism that underlies this correlation was unknown. We show here that CD30 was expressed on activated CD4+ T cells in the presence of IL-4. In the absence of endogenously produced IL-4, however, even Th2 lineage cells lost CD30 expression. Thus, CD30 is not an intrinsic marker of Th2 cells, but is inducible by IL-4. CD30 was also found to be down-regulated by IFN-gamma. Committed Th1 effector cells do not express CD30, although differentiating Th1 lineage cells temporarily express CD30. The transient expression of CD30 on differentiating Th1 lineage cells was mainly the result of endogenously produced IL-4 induced by IL-12. Culture of IL-12-primed cells under conditions that reverse the phenotype (Ag plus IL-4) resulted in two cell populations based upon their ability to express CD30. One population responded to IL-4 upon restimulation and became a CD30-positive, Th0-like cell population, while the other remained CD30 negative and synthesized only IFN-gamma. Thus, CD30 expressed on CD4+ T cells reflected the ability of CD4+ T cells to respond to IL-4.
Monoclonal antibodies specific for beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells.
Authors: Picarella D , Hurlbut P , Rottman J , Shi X , Butcher E , Ringler D J
Published: December 2022

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is an adhesion protein expressed on endothelium in mucosal tissues that has been shown to play an important role in the selective homing of lymphocytes to intestinal mucosa and associated lymphoid tissue. To determine whether MAdCAM-1 or its ligand alpha 4 beta 7 would be appropriate targets for therapeutic intervention in gut-associated inflammation, we tested the ability of rat mAbs specific for beta 7 integrin and MAdCAM-1 to inhibit chronic colonic inflammation in scid mice reconstituted with CD4+ T cells enriched for the CD45RBhigh subpopulation. Abs specific for beta 7 and MAdCAM-1 blocked recruitment of lymphocytes to the colitic colon, and more importantly, these Abs significantly reduced the severity of colonic inflammatory disease in this animal model. Therefore, the adhesive interactions mediated by alpha 4 beta 7 and MAdCAM are intimately involved in leukocyte recruitment to gut in chronic inflammatory disease and may be relevant therapeutic targets for patients with inflammatory bowel disease.
MUC18/MCAM (CD146), an activation antigen of human T lymphocytes.
Authors: Pickl W F , Majdic O , Fischer G F , Petzelbauer P , Faé I , Waclavicek M , Stöckl J , Scheinecker C , Vidicki T , Aschauer H , Johnson J P , Knapp W
Published: December 2022

Extravasation and tissue infiltration of leukocytes and metastatic tumor cells require the regulated expression and function of adhesive and pro-proteolytic surface molecules. We demonstrate here that human T cells, upon activation, neo-express the melanoma metastasis-associated surface molecule MUC18/melanoma cell adhesion molecule (MCAM). Expression of MUC18/MCAM (CD146) on T cells could be identified with two mAbs (541-10B2 and 541-2E5) obtained after immunization with HUT102 T cells and found to react with activated T cells. The specificity of our mAbs for MUC18/MCAM (CD146) was revealed by 1) definition of the appropriate molecular mass of approximately 110 kDa unreduced and 120 kDa reduced, 2) reactivity of mAbs with MUC18/MCAM (CD146) cDNA-transfected mouse L cells, 3) conclusive crosswise immunoblotting experiments with MUC18/MCAM (CD146)-specific mAbs, and 4) N-terminal amino acid sequencing of precipitated protein. In vitro activation by PHA caused neo-expression of MUC18/MCAM (CD146) on peripheral blood T cells within 1 day of stimulation, reaching a maximum on day 3. In vivo expression of MUC18/MCAM (CD146) was confirmed on CD3+ T cells infiltrating delayed-type hypersensitivity lesions of the skin, on synovial fluid T cells of rheumatoid arthritis patients, and on distinct T leukemia cells. MUC18/MCAM (CD146) cell surface expression on activated T cells is mirrored by the presence of specific mRNA. Leukocytes of healthy donors do not show significant MUC18/MCAM (CD146) expression. The finding that MUC18/MCAM (CD146) is also expressed on activated T cells might suggest that this adhesion molecule is involved in the extravasation and/or homing of activated T cells.
The nonpolymorphic MHC class II isotype, HLA-DQA2, is expressed on the surface of B lymphoblastoid cells.
Authors: Rudy G B , Lew A M
Published: December 2022

The more centromeric of the two human MHC class II DQA genes, DQA2, has not previously been shown to express a protein product. However, its high degree of sequence similarity to DQA1, the fact that it is highly evolutionarily conserved, and the recent demonstration of its transcription in some cell lines suggest that a DQA2 alpha-chain may be expressed. Polyclonal anti-peptide antisera were generated and shown to be specific for DQA2 in Western blotting of Triton X-114 preparations of B lymphoblastoid cell lines. DQA2 expression is variable, although always at least threefold less than that of DQA1, and is found in all haplotypes examined. Immunoprecipitations of biotin-labeled cell surface proteins reveal that DQA2 appears at the cell surface. In keeping with others' findings, no evidence for an expressed DQB2 beta-chain was found, prompting investigation of the means by which DQA2 gains access to the cell surface. Antisera specific for allotypic and isotypic MHC class II beta-chains were used in sequential immunoprecipitation experiments to search for a beta-chain partner for DQA2, and a transfectant system was established in an attempt to promote pairing of DQA2 with a selected DQB1 (*0501) chain. In neither case was DQA2 found to dimerize with an MHC class II beta-chain. Despite this, DQA2 is capable of forming a complex with invariant chain.
Both innate and acquired immunity to Listeria monocytogenes infection are increased in IL-10-deficient mice.
Authors: Dai W J , Köhler G , Brombacher F
Published: December 2022

IL-10-deficient mice were highly resistant to Listeria monocytogenes during the course of infection. An increased innate immunity was suggested by reduced bacterial burdens (as much as 50-fold) early (days 2 and 3) in the infection, as compared with control mice. In addition, in vitro stimulation of both IL-10-deficient peritoneal exudate cells and spleen cells with heat-killed Listeria resulted in a dramatically enhanced proinflammatory cytokine response (e.g., IL-12, IFN-gamma, TNF-alpha, IL-1alpha, and IL-6). During later stages of a primary Listeria infection, the reduced bacterial burden in the infected organs of IL-10-deficient mice was accompanied by decreased tissue damage and earlier clearance of the pathogen, as well as a stronger Th1 polarization. The absence of IL-10 did not influence membrane-bound factors that stimulate Th cell responses, demonstrated by the finding of normal MHC class II, B7.1, and B7.2 surface expression on F4/80+ macrophages in vivo. IL-10-deficient mice were also more resistant to a secondary infection, accompanied by an enhanced Th1 response. The results presented in this work demonstrate that the absence of IL-10 augments innate and acquired immunity during primary and secondary L. monocytogenes infection by up-regulating proinflammatory type 1 cytokine responses. The resulting protective Th1 responses lead to an effective reduction of bacterial growth and tissue destruction and to an earlier clearance of the bacteria. The physiologic role of IL-10 during L. monocytogenes infection studies is discussed and compared with pathogenic infections that induce a more systemic cytokine response in IL-10-deficient mice.
The role of the CD28/B7 interaction in the regulation of NK cell responses during infection with Toxoplasma gondii.
Authors: Hunter C A , Ellis-Neyer L , Gabriel K E , Kennedy M K , Grabstein K H , Linsley P S , Remington J S
Published: December 2022

We examined the role of the CD28/B7 interaction in regulation of NK cell activity. Cells transfected with B7 enhanced IL-12-induced production of IFN-gamma by IL-2-activated, CD28+ NK cells, but not by resting CD28- NK cells. The ability of B7 transfectants to enhance NK cell production of IFN-gamma was dependent on the intracellular adhesion molecule-1/LFA-1 interaction and could be inhibited by TGF-beta, but not IL-10. Since IL-12-induced production of IFN-gamma by NK cells is associated with resistance to certain infections, we examined whether the CD28/B7 interaction regulated NK cell responses during infection. Infection of SCID mice with Toxoplasma gondii resulted in the appearance of a population of CD28+ NK cells, NK cell production of IFN-gamma, and increased NK cell cytolytic activity. Administration of CTLA4-Ig to SCID mice infected with T. gondii inhibited these latter two effects and resulted in a significant increase in parasite burden. The stimulus for CD28 expression by NK cells in SCID mice infected with T. gondii appeared to be independent of IL-2. However, mRNA for IL-15, a cytokine with properties similar to those of IL-2, was detected in tissues of SCID mice infected with T. gondii. In vitro experiments demonstrated that IL-15 could stimulate resting NK cells to express functionally active CD28 as well as enhance the production of IFN-gamma by SCID splenocytes stimulated with T. gondii. Together our data demonstrate that the interaction of CD28+ NK cells with B7 regulates NK cell production of IFN-gamma associated with resistance to infection and that IL-15 may be involved in these events.
Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils.
Authors: Kitayama J , Carr M W , Roth S J , Buccola J , Springer T A
Published: December 2022

At inflammatory sites in vivo, leukocytes may confront multiple, competing chemoattractive signals. We found significant differences between eosinophils and neutrophils in transendothelial chemotaxis to a chemoattractant diffusing from the lower chamber, when a chemoattractant that binds to another receptor is present at uniform concentration. The transendothelial migration of eosinophils to FMLP, C5a, RANTES, or MCP-3 was totally inhibited by the presence of the homologous chemoattractant, and only RANTES and MCP-3 showed mutual inhibition. C5a and to a lesser extent FMLP chemokinetically stimulated migration to RANTES and MCP-3, without stimulating random migration. Results with neutrophils contrasted. The presence of FMLP not only abrogated neutrophil transmigration to FMLP but also strongly decreased chemotaxis to C5a, IL-8, and Gro-alpha. Similarly, C5a inhibited neutrophil chemotaxis to IL-8 and Gro-alpha. IL-8 almost totally abrogated chemotaxis to Gro-alpha, but Gro-alpha only moderately inhibited chemotaxis to IL-8. Neither IL-8 nor Gro-alpha significantly inhibited transmigration to FMLP or C5a. Actin polymerization in eosinophils and neutrophils was desensitized by the same combinations of chemoattractants that desensitized chemotaxis. We conclude that eosinophils have at least three noninterfering receptor-signal transduction pathways for chemotaxis and actin polymerization. In contrast, the signaling pathways for FMLP, C5a, and IL-8/Gro-alpha in neutrophils are heterologously cross-desensitized, with a hierarchy of resistance to competing signals of FMLP > C5a > IL-8 > Gro-alpha, in agreement with previous results in neutrophils on the Ca2+-mobilizing response. These results may have important implications for the behavior of these cell types in inflammatory sites.
Impaired tyrosine phosphorylation and Ca2+ mobilization, but not degranulation, in lyn-deficient bone marrow-derived mast cells.
Authors: Nishizumi H , Yamamoto T
Published: December 2022

Signaling through the high affinity IgE receptor (Fc epsilon RI) on mast cells and basophils results in rapid increases in tyrosine phosphorylation on a number of proteins. Fc epsilon RI associates with two classes of the tyrosine kinases, the Src family kinases, such as Lyn, c-Yes, and c-Src, and the Syk kinase. In this work, using primary mast cells derived from wild-type (lyn +/+) and lyn-deficient (lyn -/-) mice, we report that Lyn plays a part in signaling via Fc epsilon RI. Unlike lyn +/+ mast cells, cross-linking of Fc epsilon RI in lyn -/- mast cells failed to induce protein-tyrosine phosphorylation of various substrates, and evoked a delayed and slow Ca2+ mobilization. However, degranulation, adhesion, and production of cytokines occurred normally in lyn -/- mast cells. Our data suggest that the activity of the other Src family kinases, such as c-Src, can complement the role of Lyn in inducing most, but not all, biologic and biochemical responses to Fc epsilon RI cross-linking.
Nitric oxide, an autocrine regulator of wound fibroblast synthetic function.
Authors: Schäffer M R , Efron P A , Thornton F J , Klingel K , Gross S S , Barbul A
Published: December 2022

Nitric oxide (NO) is synthesized in wounds, but its exact role and cellular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hypothesized that WF may be also phenotypically altered during wound healing to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferation in response to 10% fetal bovine serum was assessed by [3H]thymidine incorporation in a microculture system. A fibroblast-populated collagen lattice was used for assaying contractility. Collagen synthesis was determined by measuring the collagenase-sensitive fraction of protein-incorporated [3H]proline. Fibroblasts were incubated in the presence or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monomethyl-L-arginine, both competitive inhibitors of NO synthase. WF spontaneously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/microg DNA/48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesis was limited to the first and second passages postharvest and was inhibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridization and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediated collagen contraction was enhanced (p < 0.01), and collagen synthesis was significantly decreased (p < 0.05) by inhibiting NO synthase. The data show that WF are phenotypically altered during the healing process to synthesize NO, which, in turn, regulates their collagen synthetic and contractile activities.
Activation of the phosphatidylinositol 3-kinase serine kinase by IFN-alpha.
Authors: Uddin S , Fish E N , Sher D A , Gardziola C , White M F , Platanias L C
Published: December 2022

During engagement of the type I IFN receptor, IRS-1 is phosphorylated on tyrosine and associates with the p85 regulatory subunit of the phosphatidylinositol (PI) 3'-kinase, which is a dual-specificity enzyme possessing both lipid and serine kinase activities. We sought to determine whether treatment of cells with IFN-alpha activates the PI 3'-kinase serine kinase. 32P-labeling experiments and phosphoaminoacid analysis of immunoprecipitated IRS-1 protein demonstrated that, in addition to tyrosine phosphorylation, IFN-alpha induces its phosphorylation on serine residues. In vitro kinase assays on alphaIRS-1 immunoprecipitates also demonstrated IFN-alpha-dependent serine phosphorylation of IRS-1, suggesting that the protein associates with an IFN-alpha-regulated serine kinase. Furthermore, IFN-alpha-dependent phosphorylation of IRS-1 was detected in in vitro kinase assays on alpha p85 immunoprecipitates, and was inhibited by pretreatment of cells with the specific PI 3'-kinase inhibitor wortmannin, consistent with a regulatory role of the PI 3'-kinase serine kinase on the phosphorylation of the protein. Treatment of cells with wortmannin also inhibited the phosphorylation of the p85 subunit of PI 3'-kinase and the type I IFN-regulated activation of the Map kinase, but had no inhibitory effect on the IFN-alpha-induced activation of Tyk-2 and Jak-1 kinases nor on the activation of Stat-1, Stat-2, and Stat-3. Taken all together, these data establish that the PI 3'-kinase serine kinase is activated by IFN-alpha and may play an important role in the transmission of type I IFN receptor-generated signals.
Enhancement of TNF receptor p60-mediated cytotoxicity by TNF receptor p80: requirement of the TNF receptor-associated factor-2 binding site.
Authors: Weiss T , Grell M , Hessabi B , Bourteele S , Müller G , Scheurich P , Wajant H
Published: December 2022

TNFR p60 (TNFR60) is the main mediator of TNF-induced apoptosis, although in some cells TNFR80 is also involved. TNFR80-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, "ligand passing," or induction of endogenous TNF. Using HeLa transfectants expressing wild-type TNFR80 and deletion mutants derived from them, we have investigated the mechanism of TNFR80-mediated cytotoxicity. TNFR80 induces no cytotoxicity on its own, but is functional in these transfectants, as selective stimulation activates nuclear factor-kappaB (NF-kappaB) and induces NF-kappaB-dependent cellular responses (IL-6 and manganese superoxide dismutase). TNFR60-induced cytotoxicity, however, is enhanced about 1000-fold by costimulation of TNFR80, whereas only additive responses are observed with regard to NF-kappaB-dependent responses. Ligand passing is not critically involved, because a similar potentiation of TNFR60-mediated cytotoxicity was observed when agonistic Abs were used for stimulation of both receptors. In addition, in HeLa cells overexpressing a truncated form of TNFR80 lacking the cytoplasmic domain, no TNFR80-dependent potentiation of TNFR60-mediated cytotoxicity was detectable, emphasizing that intracellular signaling of TNFR80 is required for synergistic activity. The involvement of endogenously produced TNF can be ruled out by the use of TNF-neutralizing Abs. The ability of TNFR80 to cooperate with TNFR60 could be mapped to the binding site for the TNF receptor-associated factor-2. These results are in agreement with a differential cooperation of both TNFR; an additive effect is obtained in those responses that are initiated by both receptors via identical pathways, i.e., activation of NF-kappaB, whereas induction of cytotoxicity is most likely mediated by distinct signaling pathways, allowing positive cooperativity.
Proinflammatory cytokines inhibit HIV-1(SF162) expression in acutely infected human brain cell cultures.
Authors: Lokensgard J R , Gekker G , Ehrlich L C , Hu S , Chao C C , Peterson P K
Published: December 2022

An understanding of how viral replication in glial cells responds to proinflammatory cytokines is important in delineating HIV-1 neuropathogenesis. Because no information is available in the literature regarding the regulatory effects of exogenous cytokines on acute HIV-1 replication in human brain cells, we studied the impact of cytokine treatment on viral p24 Ag expression. Based upon reports using mononuclear phagocytes derived from somatic sources, we hypothesized that TNF-alpha, IL-1 beta, and IL-6 would up-regulate the expression of HIV-1(SF162) (a monocytotropic strain) in purified microglial cells and in mixed brain cell cultures. This hypothesis was not supported. In fact, a contrary, unexpected result was obtained; whereas in purified microglial cultures TNF-alpha displayed a mild stimulatory effect on HIV-1 expression (15% increase in p24 Ag production compared with control cultures), surprisingly, IL-1 beta and IL-6 were highly suppressive (91 and 83% inhibition of HIV expression, respectively). In contrast to the findings in microglial cell cultures, TNF-alpha profoundly suppressed (84%) HIV-1 expression in mixed brain cell cultures, as did IL-1 beta (82%), and IL-6 was moderately suppressive (55% inhibition). In an attempt to identify factors responsible for the differential effects of TNF-alpha in the two brain cell infection models, it was found that compared with microglial cell cultures, TNF-alpha treatment of mixed brain cell cultures released significantly greater amounts of RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1 alpha, beta-chemokines that have been suggested to have anti-HIV-1 effects. Thus, these data suggest that proinflammatory cytokines possess anti-HIV-1 activity in the central nervous system.
VHDJH gene sequences and antigen reactivity of monoclonal antibodies produced by human B-1 cells: evidence for somatic selection.
Authors: Schettino E W , Chai S K , Kasaian M T , Schroeder H W , Casali P
Published: December 2022

To understand whether the distinct VHDJH gene utilization by natural polyreactive Abs reflects the developmentally restricted Ig VHDJH rearrangements putatively expressed by B-1 cells, we generated 11 (8 IgM, 1 IgG3, 2 IgA1), 7 (6 IgM, 1 IgG1), and 7 (2 IgM, 3 IgG1, 2 IgG3) mAb-producing lines using B-1a (surface CD5+, CD45RAlow), B-1b (surface CD5-, CD45RAlow, CD5 mRNA+), and B-2 (surface CD5-, CD45RAhigh, CD5 mRNA-) cells, respectively, sorted from adult human peripheral blood. Most B-1a and B-1b, but no B-2, cell-derived mAbs were polyreactive; i.e., they bound different self and foreign Ags with different affinities. B-1a and B-2 mAbs preferentially utilized VH4 (p = 0.003) and VH3 (p = 0.010) genes, respectively. All three mAb populations utilized DXP, DLR, DN DH genes, and JH6, but no mAb utilized DHQ52. There were fewer unencoded nucleotide (N) additions in the VHDJH junctions of B-1b (3.00 +/- 2.52, mean +/- SD) than of B-1a (12.45 +/- 3.93, p = 1.23 x 10(-5)) or B-2 (8.29 +/- 4.75, p = 0.020) mAbs. Partly due to the fewer N additions and a paucity of D-D fusions, the B-1b mAb CDR3s were significantly shorter than the B-1a mAb CDR3s (p = 0.013), which contained a nonrandom Tyr distribution (p = 0.003). Finally, all but two B-1 cell-derived mAbs were mutated, in a fashion similar to that of the Ag-selected B-2 mAbs. Thus, in the human adult, B-1 cells that make natural polyreactive Abs may not be representative of the predominantly B-1 developmental waves of colonization of the fetal and neonatal B cell repertoires, and are somatically selected.