bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide induces synthesis of mouse colony-stimulating factor-1 in vivo.
Authors: Roth P , Bartocci A , Stanley E R
Published: December 2022

CSF-1 is a hemopoietic growth factor that regulates the survival, proliferation, and differentiation of mononuclear phagocytes, cells that are critical in the inflammatory response. In the case of Gram-negative infection, LPS plays an important role by inducing several cell types to produce the proinflammatory cytokines, IL-1, IL-6, and TNF-alpha. In this study, we examined the effects of i.p. administration of LPS on CSF-1 expression in the mouse. Two- to sevenfold increases in the CSF-1 concentrations determined by RIA were evident within hours of LPS administration in serum, liver, kidney, lung, spleen, brain, intestine, and heart. While alterations in the CSF-1 receptor-mediated clearance of CSF-1 appeared not to account for the increased growth factor concentrations in LPS-treated animals, there was an early LPS-induced reduction of splenic [125I]CSF-1 uptake consistent with tissue-specific down-modulation of CSF-1 receptors. The results of Northern analysis revealed increased expression of a CSF-1 mRNA species in liver, lung, kidney, spleen, intestine, and heart following LPS treatment, demonstrating that increased synthesis was responsible for the increased tissue CSF-1 concentrations. The increased expression and synthesis of CSF-1 in response to LPS may be essential for mobilizing and activating mononuclear phagocytes in the inflammatory response.
A single amino acid in the p58 killer cell inhibitory receptor controls the ability of natural killer cells to discriminate between the two groups of HLA-C allotypes.
Authors: Winter C C , Long E O
Published: December 2022

To examine the structural basis for the specific recognition of the MHC class I allotypes HLA-Cw*0401 and HLA-Cw*0304 by the killer cell inhibitory receptors (KIR) cl42 and cl43, respectively, mutant KIR-Ig fusion proteins were tested by direct binding to cells transfected with single HLA-C alleles. The putative loop region at position 44-46 of KIR contained amino acids that were necessary for the discrimination between HLA-Cw*0401 and HLA-Cw*0304. Surprisingly, exchanging the methionine at position 44 in cl42 with the lysine at position 44 in cl43 was sufficient to switch the specificity of cl42 from HLA-Cw*0401 to HLA-Cw*0304, and vice versa. Thus, a single amino acid in the first Ig domain of these KIR determines their ability to discriminate between the two groups of HLA-C allotypes.
Disruption of T cell tolerance by directing a self antigen to macrophage-specific scavenger receptors.
Authors: Abraham R , Choudhury A , Basu S K , Bal V , Rath S
Published: December 2022

Breakdown of immune self tolerance is speculated to cause autoimmune diseases, but most studies on tolerance use foreign molecules as targets. In this study, we show another approach using delivery of a maleylated self protein to macrophage-specific scavenger receptors. Mice generate Abs against the maleylated form of a ubiquitous self Ag, mouse serum albumin (MSA), although native MSA is nonimmunogenic. This generation of anti-maleyl MSA Abs depends on binding of maleyl MSA to scavenger receptors in vivo, since coinjection of a serologically unrelated scavenger receptor ligand inhibits it, suggesting that the Ab response is T cell dependent. Spleen cells as well as nylon adherence-purified splenic T cells from maleyl MSA-immune mice proliferate in response to both maleyl MSA and MSA; this response is blocked by anti-MHC class II mAbs, and the autoimmune cells can recognize at least five 15-mer peptides from the MSA sequence, establishing that T cell tolerance to MSA has been broken in these mice. Maleyl MSA and MSA are recognized equally well, provided the scavenger receptor-specific delivery of maleyl MSA is blocked during stimulation in vitro, indicating that maleyl MSA-specific non-self peptides are unlikely to play a major role in the observed disruption of T cell tolerance. Thus, delivery of some self molecules to scavenger receptors may lead to disruption of immune tolerance. These results are relevant to mechanisms of immune tolerance and the etiopathogenesis of autoimmunity.
Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines.
Authors: Beatty P R , Krams S M , Martinez O M
Published: December 2022

Immunocompromised individuals have an increased incidence of EBV-associated B cell lymphomas. The growth factors responsible for the unrestrained proliferation of these lymphomas have not yet been determined. In this study, spontaneous lymphoblastoid cell lines (SLCL) were derived without the addition of growth factors or virus from four patients with EBV-associated lymphoproliferative disorder. These cell lines were EBV transformed in vivo, and infection was verified through amplification of the viral gp220 gene. SLCL have an activated B cell phenotype (CD19+, CD21+, CD23+, CD38+, and CD40+) and produce IL-6, IL-10, TNF-alpha, and lymphotoxin-alpha. To determine whether these cytokines contribute to autonomous growth, neutralizing Abs for IL-10, IL-6, and TNF-alpha, a soluble TNFR:Fc fusion protein, and soluble IL-10R were used. These experiments established that, of the cytokines produced by SLCL, only IL-10 is an autocrine factor. IL-10 was produced by the majority of cells within each SLCL, and IL-10 secretion was concomitant with SLCL growth. Our findings demonstrate that IL-10 is utilized in the autonomous growth of EBV-related lymphomas and may be crucial in the development of lymphoproliferative disorder.
Distribution of Ly-6C on lymphocyte subsets: I. Influence of allotype on T lymphocyte expression.
Authors: Schlueter A J , Malek T R , Hostetler C N , Smith P A , deVries P , Waldschmidt T J
Published: December 2022

The expression patterns of the Ly-6C Ag were examined on splenic and thymic lymphocyte subsets of Ly-6.1 and Ly-6.2 strains of mice using the rat mAb 15.1. Ly-6C is expressed on subsets of CD4+ and CD8+ splenocytes, and a portion of NK cells. Within the splenic and lymph node CD4+ T cell compartment, Ly-6C expression is restricted to Ly-6.2 strains of mice, and is present on a subset of naive cells. Ly-6C is expressed on the majority of peripheral CD8+ T cells in both Ly-6.1 and Ly-6.2 strains, and is found primarily on the Ag-experienced subset. In the thymus, Ly-6C is present on subpopulations of CD4- CD8+, CD4- CD8-, and CD4+ CD8- cells. Ly-6C+ CD4- CD8+ thymocytes show a mature phenotype, while Ly-6C+ CD4- CD8- and Ly-6C+ CD4+ CD8- thymocytes appear to be part of the recently described NK1.1+ alphabeta TCR+ population. On account of the marked differences in Ly-6C expression on peripheral CD4+ T cells from Ly-6.1 and Ly-6.2 strains of mice, additional experiments were undertaken to assess Ly-6C expression in parental and Ly-6.1 x Ly-6.2 F1 mice. Neither phenotype dominated in the F1 offspring, with frequencies of Ly-6C+ CD4+ splenocytes falling in the intermediate range. Further experiments compared the staining patterns of the rat anti-pan Ly-6C (Ly-6.1 and Ly-6.2) Ab with a mouse anti-Ly-6.2 allotype specific Ab, with emphasis on both Ly-6.2 and Ly-6.1 x Ly-6.2 F1 mice. The results demonstrate the presence of lymphocytes that express the pan form of Ly-6C but not the form recognized by the alloantibody. This latter finding suggests the presence of more than one form of the Ly-6C Ag.
V(H) repertoire maturation during B cell development in vitro: differential selection of Ig heavy chains by fetal and adult B cell progenitors.
Authors: Marshall A J , Paige C J , Wu G E
Published: December 2022

B cell development is characterized by marked changes in Ig repertoire, which include shifts in the pattern of V(H) segment usage. B cell precursors characteristically utilize a restricted set of V(H) segments, while mature B cell populations use a wide range of V(H) segments. V(H)81x is an example of a V(H) segment that is highly utilized in B cell precursors, but is rarely utilized in mature B cells. To dissect the molecular and cellular requirements for Ig repertoire maturation, we have examined V(H)81x usage in an in vitro model of B cell development. We find that primary fetal or adult B cell progenitors differentiating in vitro mimic progenitors differentiating in vivo with respect to V(H)81x overusage and subsequent decline in V(H)81x usage, showing that neither of these events is dependent on the intact architecture of the primary lymphoid organ or contact with stromal cells. The relative decline in V(H)81x usage in cultures initiated with adult progenitors was associated with a decrease in the ratio of productive/nonproductive V(H)81x-DJ(H) rearrangements; however, an increase in this ratio was observed in identical cultures initiated with fetal progenitors. This result indicates a difference in selection of V(H)81x-encoded heavy chains that is intrinsic to fetal and adult B cell progenitors. Thus, while the relative decline in V(H)81x usage during adult development can be at least partially explained by selection against cells bearing V(H)81x-encoded heavy chains, other mechanisms must be postulated to explain the decline in V(H)81x usage during fetal development.
Heat shock protein 72 on tumor cells: a recognition structure for natural killer cells.
Authors: Multhoff G , Botzler C , Jennen L , Schmidt J , Ellwart J , Issels R
Published: December 2022

Evidence is accumulating that members of the heat shock protein 70 (HSP70) family are found on the cell surface of certain tumor cells where they elicit a strong antitumor immune response. We demonstrated that HSP72, the major heat-inducible form of the HSP70 group, is located on the cell surface of approximately 60% of the human colon carcinoma cells CX2 with two different mAbs by indirect immunofluorescence, by electron microscopy, and by selective cell surface biotinylation. In an effort to analyze the role of HSP72 cell surface expression as a tumor-specific recognition structure within an "autologous" tumor system, the CX2 cells were separated into a stably HSP72 high expressing (CX+: >90%) and a stably HSP72 low expressing (CX-: <20%) subline. The expression "autologous" was written in parentheses to indicate that the colon carcinoma sublines CX+ and CX- derived from the original CX2 tumor cell line differ with respect to the cell surface expression pattern of HSP72, whereas they exhibit an identical cell surface expression pattern of MHC and cellular adhesion molecules (e.g., intercellular cellular adhesion molecule, neural cellular adhesion molecule, vascular cellular adhesion molecule). Within this "autologous" tumor cell system, we demonstrate that the sensitivity to lysis mediated by adherent non-MHC-restricted effector cells correlates (p < 0.05) with the amount of HSP72 that is expressed on the cell surface. Blocking studies using an HSP72-specific mAb revealed that HSP72 might act in an MHC-unrestricted manner as a tumor-specific recognition structure for a distinct NK cell population.
Properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils.
Authors: Wirthmueller U , Dewald B , Thelen M , Schäfer M K , Stover C , Whaley K , North J , Eggleton P , Reid K B , Schwaeble W J
Published: December 2022

Properdin is an important regulatory constituent of the complement system. In contrast to most other components of complement, biosynthesis of properdin is restricted to a few cell types only, i.e., monocytes/macrophages and peripheral blood T cells. This report demonstrates the presence of properdin mRNA in peripheral blood granulocytes and shows that properdin is stored in the granules of human neutrophils and secreted upon stimulation with TNF-alpha, C5a, IL-8, or FMLP. Subcellular fractionation using Percoll density gradients and Western blot analyses revealed that the bulk of properdin is contained in the secondary granules. Moreover, flow cytometric analyses indicated that properdin is present on the surface of neutrophils. In contrast to alternative pathway components, components of the classical pathway of complement activation, such as C2 and C4, were not detected. Our findings suggest that neutrophils can actively stabilize and amplify the alternative activation pathway of complement by secretion of properdin as part of the innate defense against microorganisms.
Transgene-induced production of IL-4 alters the development and collagen expression of T helper cell 1-type pulmonary granulomas.
Authors: Lukacs N W , Addison C L , Gauldie J , Graham F , Simpson K , Strieter R M , Warmington K , Chensue S W , Kunkel S L
Published: December 2022

A number of complex mechanisms regulate the size and cellularity of an Ag-dependent granulomatous reaction and their accompanying cytokine production profiles. In the present study, Th1 (purified protein derivative (PPD)) and Th2 (schistosome egg Ag)-type granulomas were established to examine the role of IL-4 in lesion development and procollagen type III expression. PPD-sensitized mice were transfected via the airway with an adenovirus construct containing an IL-4 cDNA cassette, and the lungs were embolized with sized Ag-coated Sepharose beads. Granuloma development in response to the PPD bead challenge in control mice primarily consisted of mononuclear cells. In contrast, the overexpression of IL-4 in the IL-4 adenovirus-transfected animals demonstrated a significant increase in cellularity, size, procollagen type III expression, and accumulation of eosinophils. Analysis of mRNA and protein within the lung demonstrated significant expression of IL-4 in only the IL-4 adenovirus-transfected animals. The granuloma lesion size was significantly increased in the IL-4 adenovirus-transfected animals on days 2 and 5, reaching an approximate 50% increase compared with the control groups. Furthermore, procollagen type III mRNA expression was increased in the IL-4 adenovirus-transfected, PPD bead-embolized lungs. In contrast, when IL-4 was neutralized during Th2-type schistosome egg Ag bead granulomas, a decrease in procollagen type III was observed. These data indicate that the progression of certain granulomas may be regulated by the production of IL-4, thus altering the cellularity, size, and matrix composition of the inflammatory response.
A noncytolytic IL-10/Fc fusion protein prevents diabetes, blocks autoimmunity, and promotes suppressor phenomena in NOD mice.
Authors: Zheng X X , Steele A W , Hancock W W , Stevens A C , Nickerson P W , Roy-Chaudhury P , Tian Y , Strom T B
Published: December 2022

We have been successful in our efforts to develop a long lived noncytolytic murine IL-10/Fc fusion protein. In the nonobese diabetic mouse (NOD) model, administration of IL-10/Fc from 5 to 25 wk of age completely prevented the occurrence of diabetes. Moreover, these mice remained disease-free long after cessation of IL-10/Fc therapy. Immunohistochemistry studies show that IL-10/Fc treatment inhibits expression of TNF-alpha, proinflammatory cytokine, as well as Th1-type cytokines, IL-2 and IFN-gamma, but promotes expression of IL-4 and IL-10, Th2-type cytokines, by islet-infiltrating leukocytes. In an adoptive transfer model of diabetes in NOD mice, we found that: 1) IL-10/Fc treated hosts bear leukocytes that block expression of diabetes and 2) these leukocytes persisted even 8 wk after cessation of IL-10/Fc treatment. The potent antidiabetogenic effects provided by IL-10/Fc in the NOD model, together with its apparent lack of systemic toxicity, are notable.
Inhibition of primary and recall allergen-specific T helper cell type 2-mediated responses by a T helper cell type 1 stimulus.
Authors: Scott D E , Agranovich I , Inman J , Gober M , Golding B
Published: December 2022

Allergic responses are characterized by the production of Ag-specific IgE Abs that are dependent upon Th2-mediated T cell help. We determined whether heat-killed Brucella abortus (BA), an inducer of Th1 responses, could influence the allergic Th2-mediated IgE response to OVA adsorbed to alum (O/A). BA plus O/A, but not O/A alone, induced high levels of mRNA for IFN-gamma and IL-12 promptly after injection. Furthermore, initial treatment with BA plus O/A rendered both BALB/c and C57Bl/6 mice incapable of mounting high IgE responses even after repeated challenges with allergen alone. Long term abrogation of anti-OVA IgE correlated with an increased frequency of IFN-gamma-secreting OVA-specific cells and a decreased frequency of IL-4-secreting OVA-specific cells. Initial treatment with anti-IL-12 prevented BA-induced early IFN-gamma production and secondary IgG2a responses, but did not abrogate IgE suppression. Additionally, secondary OVA-specific IgE responses were down-regulated by BA conjugated to OVA or by BA given with O/A. BA-induced down-regulation of secondary IgE responses was associated with increased frequency of Ag-specific IFN-gamma-secreting cells. These results suggest the possibility that even recall Th2-mediated immune responses can be attenuated if Ag is given with a carrier or adjuvant that induces potent Th1-promoting cytokines.
Bcl-2 expression in target cells leads to functional inhibition of caspase-3 protease family in human NK and lymphokine-activated killer cell granule-mediated apoptosis.
Authors: Renvoizé C , Roger R , Moulian N , Bertoglio J , Bréard J
Published: December 2022

In the granule exocytosis pathway of cell-mediated cytotoxicity, rapid apoptotic nuclear damage in target cells has been unequivocally linked to granzyme B activity. Direct cleavage and activation of caspase-3 and related proteases by granzyme B have been identified as a central event in apoptosis induction by cytotoxic granules. The Bcl-2 oncoprotein has been recently shown to act at the level or upstream of caspase-3 family activation to inhibit apoptosis induced by various stimuli including Fas ligation, an alternative cell-mediated lytic pathway. In this study, we have investigated whether activation of this caspase family by granzyme B, during human NK and lymphokine-activated killer cell granule-mediated apoptosis, could be influenced by Bcl-2 expression. Bcl-2-overexpressing clones were generated from parental K562 and U937 cell lines (K6 and U4 clones, respectively). Bcl-2 expression abrogated early 125I-DNA release and DNA fragmentation, these defects being compensated for by extended incubation times. Cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 family substrate, was detected in parental K562 cells exposed to lymphokine-activated killer effectors but not in K6 targets, indicating that caspase-3 and related proteases function was inhibited by Bcl-2. Functional inhibition of caspase-3 family with benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe) fluoromethylketone led to similar consequences on apoptotic nuclear events as for Bcl-2 expression. Thus, Bcl-2 antagonizes granzyme B-mediated apoptosis by a mechanism that interferes with caspase-3 activity. Finally, Bcl-2 expression or the Asp-Glu-Val-Asp peptide was much less efficient in preventing phosphatidylserine externalization, suggesting that despite impaired nuclear apoptosis, immediate recognition and elimination of Bcl-2-expressing cells by tissue phagocytes should remain partly unaffected.
Ca2+-dependent, Fas- and perforin-independent apoptotic death of allografted tumor cells by a type of activated macrophage.
Authors: Yoshida R , Sanchez-Bueno A , Yamamoto N , Einaga-Naito K
Published: December 2022

Both perforin- and Fas ligand (FasL)-deficient CTLs show impaired lytic activity toward most target cells. In this study, we examined whether these molecules could be involved in the cell-to-cell contact-dependent cytotoxicity mediated by macrophages infiltrating into the rejection site of allografted Meth A fibrosarcoma cells. FasL-expressing lymph node cells from MRL-lpr/lpr mice were inactive toward Meth A tumor cells. In C3H/HeJ-gld/gld (abnormal FasL) mice, allograft-induced macrophages (AIM) were cytotoxic against Meth A cells expressing no Fas Ag. Furthermore, allografted Meth A tumor cells were acutely rejected by both C3H/HeJ-gld/gld and control C3H/HeJ mice, indicating that the cytotoxic activity of AIM against Meth A tumor cells was Fas/FasL independent. On the other hand, the cytotoxic activity of AIM against the allografts was dose dependently inhibited by EGTA; and the suppression was restored by the addition of Ca2+, but not Mg2+, implying the involvement of perforin in the cytotoxicity. In the perforin-deficient mice, however, AIM were cytotoxic against Meth A tumor cells; and allografted Meth A tumor cells were acutely rejected by both perforin-deficient and control B6 mice, indicating a perforin-independent cytotoxicity. Thus, through a yet unknown mechanism, AIM induced the apoptotic death of allografted Meth A tumor cells. These results indicate that AIM-mediated cytotoxicity against allografted Meth A tumor cells is Ca2+ dependent, and that an attack by AIM results in the apoptotic death of allografted Meth A tumor cells through a third mechanism, one other than the Fas/FasL- and perforin-based pathways.
Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish.
Authors: Ghaffari S H , Lobb C J
Published: December 2022

Earlier studies distinguished two classes of catfish light (L) chain (designated F and G). The cDNA structure and genomic organization of G L chain gene clusters has also been characterized previously. In this study, full length cDNA encoding F L chain was derived using PCR strategies based on the determined amino-terminal protein sequence. The encoded V region is readily delineated into framework regions (FR) and complementarity-determining regions (CDR). Multiple sequence alignments indicate that the F V(L) is closely related to kappa gene families. The F C(L) cannot be generally classified but it is structurally distinct from the C(L) regions of G: the amino acid sequence similarity is <35%. cDNA sequences representing processed sterile F transcripts of different loci were identified. Each sequence begins within the J(L) recombination signal sequence and extends downstream through the I(L)-C(L) segments. Genomic blots hybridized with C(L) probes indicate that there are at least 50 different C(L) segments. Based upon V(L) hybridization studies, different families of V(L) segments appear to be associated with closely related F C(L) segments. In characterized genomic clones, F gene segments are arranged in closely linked clusters with single copies of V(L), J(L), and C(L) segments within each cluster. The V(L) segments are located in opposite transcriptional polarity relative to the J(L) and C(L) segments, which indicates that V(L) segments rearrange by inversion. These combined studies establish that two structurally distinct classes of L chains are present in teleost fish and that both of the L chain classes evolved within a common organizational pattern of clustered segmental genes.
Expression of a biologically active recombinant mouse IL-1 receptor antagonist and its use in vivo to modulate aspects of the acute phase response.
Authors: Grehan S , Uhlar C M , Sim R B , Herbert J , Whitehead A S
Published: December 2022

Recombinant mouse IL-1receptor antagonist protein (rmIL-1ra) was expressed in Escherichia coli. In vivo administration of rmIL-1ra, in a casein-induced murine model of acute inflammation, completely abolished the hepatic induction of the mRNAs specifying serum amyloid A1 (A-SAA1) and A-SAA2 for up to 12 h, indicating that hepatic A-SAA mRNA synthesis is totally IL-1 driven. A-SAA protein, however, was present in the serum of rmIL-1ra-treated casein-stimulated mice (although at lower levels than in untreated casein-stimulated mice) at 12 h indicating that extrahepatic A-SAA synthesis is driven in part by factors acting independently of IL-1. Hepatic mRNA levels of the other mouse acute phase reactants (APRs), serum amyloid P component, C-reactive protein, alpha1-acid glycoprotein, and C3 were also induced with casein after 12 h, as were serum protein levels of SAP and C3. These inductions were only partially inhibited by rmIL-1ra, indicating that hepatic expression of the latter APRs (unlike that of A-SAA) is driven partly by IL-1 and partly by factors acting independently of IL-1. Hepatic mRNA levels of the negative APRs apolipoprotein A-I and serum albumin were down-regulated 12 h after casein stimulation. rmIL-1ra partially restored serum albumin mRNA levels but not apo A-I mRNA levels, indicating differential regulation of these negative APRs. The rmIL-1ra will be useful in studies of IL-1-mediated gene regulation in murine models of inflammation.
Endotoxin induces expression of type II phospholipase A2 in macrophages during acute lung injury in guinea pigs: involvement of TNF-alpha in lipopolysaccharide-induced type II phospholipase A2 synthesis.
Authors: Arbibe L , Vial D , Rosinski-Chupin I , Havet N , Huerre M , Vargaftig B B , Touqui L
Published: December 2022

Elevated levels of secretory type II phospholipase A2 (sPLA2-II) have been associated with a poor clinical outcome in the acute respiratory distress syndrome. This study identifies the cell source(s) and the mechanisms of sPLA2-II synthesis in the guinea pig model of acute respiratory distress syndrome induced by intratracheal injection of LPS. Administration of LPS led to an increase in lung membrane-associated calcium-dependent sPLA2 activity, which was abrogated by LY311727, a selective inhibitor of sPLA2-II. No sPLA2 activity was detected in the vascular compartment of the lung. LPS administration induced a parallel accumulation of sPLA2-II mRNA in lung tissues. In situ hybridization showed that sPLA2-II transcripts were synthesized in interstitial and alveolar macrophages (AM). Incubation of AM with LPS enhanced the expression of sPLA2-II mRNA, leading to stimulation of sPLA2-II synthesis and secretion. This increase was prevented by the addition of anti-TNF-alpha and anti-p55 TNF receptor Abs. Furthermore, the addition to AM of cellfree bronchoalveolar fluid collected from LPS-treated guinea pigs increased sPLA2-II expression, which was abrogated by anti-TNF-alpha Ab. These findings demonstrate that 1) macrophages are in vivo the major cell source of sPLA2-II in LPS-induced acute lung injury; 2) in contrast to that in other cell systems, regulation of LPS-induced sPLA2-II synthesis in AM is TNF-alpha dependent; and 3) production of TNF-alpha in the air-lung interface is an important step for sPLA2-II synthesis in macrophages.
Shared TCR Vbeta gene expression by the pancreas and salivary gland in immunodeficient alymphoplasic mice.
Authors: Yamamichi M , Matsuoka N , Tomioka T , Eguchi K , Nagataki S , Kanematsu T
Published: December 2022

Mice with the homozygous mutation alymphoplasia (aly) lack lymph nodes and Peyer's patches and show defects in both humoral and cellular immunity. In these mice, spontaneous infiltration of mononuclear cells was observed in multiple exocrine organs, including the pancreas, salivary glands, and lacrimal glands from the age of 15 wk, progressing to a marked tissue destruction at the age of 25 wk. Using this strain, we examined the phenotypes and TCR Vbeta gene expression of infiltrating T cells to identify the pathologic role of T cell immunity in idiopathic pancreatitis. Most of the infiltrating cells were CD4+ and Thy-1+ cells. Analysis of the TCR gene expression on T cells infiltrating the pancreas and salivary glands showed a high expression of Vbeta1 and Vbeta5 in both organs at the age of 15 wk. In contrast, a diverse expression of TCR Vbeta genes was noted at 25 wk. Sequence analysis of complementarity-determining region 3 (CDR3) of the most prominent TCR Vbeta gene family expressed in these cells, Vbeta1, showed oligoclonal expansion of infiltrating T cells in both organs. Frequent use of glutamine and proline at position 97 was observed in paired tissues. Our data suggest that oligoclonal expansion of organ specific T cells might be one of the etiologic mechanisms of chronic pancreatitis and that common autoantigens could trigger autoimmunity in multiple organs.
Spontaneously colitic C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora.
Authors: Brandwein S L , McCabe R P , Cong Y , Waites K B , Ridwan B U , Dean P A , Ohkusa T , Birkenmeier E H , Sundberg J P , Elson C O
Published: December 2022

The idiopathic inflammatory bowel diseases, ulcerative colitis and Crohn's disease, are chronic disorders that appear to arise from an aberrant interaction of environmental, genetic, and immunologic factors. The aim of this study was to examine the immune reactivity of a spontaneously colitic mouse strain, C3H/HeJBir, to epithelial, food, and enteric bacterial Ags. Serum Ab responses of colitic C3H/HeJBir and noncolitic parental C3H/HeJ mice were measured by enhanced chemiluminescence Western blotting. No reactivity to epithelial or food Ags was detected. However, the sera from C3H/HeJBir mice had a reproducible banding pattern on Western blot to bacterial Ags, whereas sera from C3H/HeJ mice did not. Only a small, highly selected number of enteric bacterial Ags were recognized. There were major differences in the degree of recognition of different bacterial strains, marked by remarkably few Abs to Ags of the major anaerobes of the bacterial flora. The serum Abs detected on immunoblot were primarily IgG2a, suggesting a Th1 response. Comparison of sera reactivity to histopathologic severity showed an inverse relationship: one third of young C3H/HeJBir mice during the peak of colitis produced Abs to bacterial Ags, while later in life, when the colitis had resolved, 96% produced Abs. These data are consistent with an abnormal immune reactivity to enteric bacterial flora in C3H/HeJBir mice, a reactivity that is highly selective considering the abundant bacterial Ags present in the colon lumen. We postulate that this reactivity plays a role in the pathogenesis of colitis in these mice.
A role for insulin-like growth factor in the regulation of IL-6-responsive human myeloma cell line growth.
Authors: Jelinek D F , Witzig T E , Arendt B K
Published: December 2022

Insulin-like growth factors (IGF-I, IGF-II) have long been recognized as important mitogens in many types of malignancies. Because the role of IGFs in growth control of myeloma cells has not been extensively examined, we have used a panel of IL-6-responsive myeloma cell lines to address this issue. Initial studies demonstrated that IGF-I and IGF-II significantly enhanced DNA synthesis by each of the four cell lines, even when assayed in the absence of IL-6. The specificity of the IGF response was confirmed using an IGF-I receptor Ab, and additional studies demonstrated that IGF responsiveness did not result from induction of autocrine IL-6 expression. When IL-6 responsiveness was assayed, three of four cell lines synthesized DNA in response to IL-6 alone; however, the magnitude of responsiveness was greatly enhanced by addition of IGFs. Similar results were obtained when proliferation and cell cycle progression were analyzed. By contrast, the KP-6 cell line was responsive to IL-6 only when IGF was present. Finally, we analyzed the effects of IGF-I on normal B lymphocytes. IGF, however, did not stimulate B cell DNA synthesis, suggesting that IGF responsiveness may represent a key difference between normal and malignant B cells. In summary, these studies suggest that IGFs may play an important role in multiple myeloma by virtue of their ability to directly stimulate tumor cell growth as well as modulate the magnitude of IL-6-driven growth.
IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early listeriosis.
Authors: Flesch I E , Wandersee A , Kaufmann S H
Published: December 2022

IL-4 is a major promotor of Th2 differentiation and an antagonist of IFN-gamma production. Although experimental listeriosis is characterized by a Th1 response, IL-4-producing cells were detected in spleens of mice promptly after Listeria monocytogenes infection. We identified this early IL-4 as inducer of the chemokine, monocyte chemoattractant protein-1 (MCP-1), which mainly attracts monocytes/macrophages, but not neutrophils. MCP-1-secreting cells were demonstrable in spleens of mice infected with L. monocytogenes, and IL-4 neutralization with anti-IL-4 mAb 11B11 markedly diminished frequencies of MCP-1-producing cells. Cell depletion experiments and studies with gene disruption mutant mice lacking distinct T cell subsets and surface MHC molecules point to CD4+ NK1+ T cells as a cellular source of early IL-4. Since monocyte infiltration to infective foci contributes to early control of listeriosis, our results suggest that IL-4-producing CD4+ NK1+ T cells participate in the innate immune response against L. monocytogenes through MCP-1 induction.
Proto-oncoprotein Vav interacts with c-Cbl in activated thymocytes and peripheral T cells.
Authors: Marengère L E , Mirtsos C , Kozieradzki I , Veillette A , Mak T W , Penninger J M
Published: December 2022

The molecular adapter c-Cbl is rapidly tyrosine phosphorylated following stimulation through the TCR and associates with Src homology domain-2 (SH2)/SH3 domain-containing adapters such as Grb2, Crk, and Crk-L, which interact with guanine nucleotide exchange factors specific for the Ras family. This suggests that c-Cbl may link TCR activation to molecules that regulate GTP binding proteins. The SH2/SH3-containing protein Vav also contains a guanine nucleotide exchange factor domain, and Vav has a crucial role in thymocyte development and activation of peripheral T cells following stimulation through the TCR. Here we show that Vav and c-Cbl form inducible molecular complexes in TCR-activated murine thymocytes and peripheral T cells as well as pervanadate-treated T cells. Vav/c-Cbl interactions are also detectable in freshly isolated T cells from gene-targeted mice that lack the T cell-specific inhibitory receptor CTLA-4, in which c-Cbl is hyperphosphorylated on tyrosine residues. The interaction between Vav and c-Cbl is directly mediated via the SH2 domain of Vav and is dependent on tyrosine phosphorylation of c-Cbl. In addition, we show that the conserved motif Y699 MTP present in c-Cbl is the binding site for the Vav SH2 domain in vitro. These data imply that c-Cbl is a molecular adapter that regulates the function of Vav in thymocytes and peripheral T cells.
Regulation of tumor antigen presentation by urocanic acid.
Authors: Beissert S , Mohammad T , Torri H , Lonati A , Yan Z , Morrison H , Granstein R D
Published: December 2022

Urocanic acid (UCA) accumulates in the epidermis after deamination of histidine. UCA isomerizes from the trans to the cis form upon exposure to environmental UV radiation. Cis-UCA is immunosuppressive in several models. Topically applied cis-UCA was reported to enhance the cutaneous tumor yield in chronically UV-irradiated mice, suggesting involvement of cis-UCA in photocarcinogenesis. Since Langerhans cells (LC) are capable of presenting tumor-associated Ags (TAA) for primary and secondary tumor-immune responses, we examined the effects of trans- and cis-UCA on LC tumor Ag presentation in a model of immunity to the S1509a spindle cell tumor (H-2a). In this system, induction of immunity requires exposure of LC to granulocyte-macrophage CSF. Naive CAF1 (H-2(a/d)) mice were immunized against S1509a by injection with granulocyte-macrophage CSF-exposed and TAA-pulsed epidermal cells (EC), as assessed by growth inhibition of inoculated tumor cells. Incubation of EC in cis-, but not trans-UCA completely inhibited Ag presentation in this system. Neither histamine antagonists nor indomethacin reversed these effects of cis-UCA. The ability of trans- and cis-UCA to modulate EC presentation of TAA for secondary immune responses was also examined. EC were pulsed with TAA in vitro and then injected into hind footpads of tumor-immune mice. After 24 h, footpad swelling was assessed as a measure of delayed-type hypersensitivity. Incubation with cis-, but again not trans-UCA before TAA exposure significantly inhibited elicitation of delayed-type hypersensitivity. These data indicate that cis-UCA may be an important regulator of LC Ag-presenting function in tumor-immune responses, and thus may play a role in photocarcinogenesis.
Propionibacterium acnes treatment diminishes CD4+ NK1.1+ T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells.
Authors: Matsui K , Yoshimoto T , Tsutsui H , Hyodo Y , Hayashi N , Hiroishi K , Kawada N , Okamura H , Nakanishi K , Higashino K
Published: December 2022

LPS injection into normal mice does not induce liver injury, while the same treatment of Propionibacterium acnes-primed mice induces severe liver injury, indicating that P. acnes treatment renders the mice susceptible to LPS. Since IFN-gamma sensitizes macrophages to LPS, we investigated the mechanism of induction and activation of IFN-gamma-producing (type 1) T cells by P. acnes. Twenty percent of liver lymphocytes of C57BL/6 mice are CD4+ NK1.1+ T cells that promptly produce IL-4 in response to anti-CD3 in vitro. However, P. acnes treatment diminished these lymphocytes. Therefore, liver lymphocytes from P. acnes-primed mice showed reduced IL-4 production. Furthermore, P. acnes treatment induced CD4- type 1 T cells in the liver. Isolated P. acnes-elicited Kupffer cells produced IL-12 and to a lesser degree IL-18 in vitro. Injection of anti-IL-12 Ab totally abrogated these actions of P. acnes, while injection of anti-IL-18 Ab caused only partial abrogation. Thus, administration of P. acnes diminished CD4+ NK1.1+ T cells, but induced type 1 T cells in the liver by induction of IL-12 and IL-18 production. Injection of IL-12 (approximately 1,000 ng) dose dependently diminished CD4+ NK1.1+ T cells, but induced type 1 T cells. In contrast, injection of IL-18 (approximately 1,000 ng) failed, although injection of a much larger dose of IL-18 (10,000 ng) or IL-18 (approximately 1,000 ng) with suboptimal doses of IL-12 (1-100 ng) diminished CD4+ NK1.1+ T cells in a dose-dependent manner. Thus, P. acnes treatment renders the mice highly susceptible to LPS by induction and activation of type 1 T cells.
New evidence for two MHC class II-restricted antigen presentation pathways by overexpression of a small G protein.
Authors: Briken V , Lankar D , Bonnerot C
Published: December 2022

Exogenous Ags may be presented by MHC class II molecules through two distinct pathways distinguished by their sensitivity to drugs that inhibit the protein synthesis. Using this approach, we previously showed that the subunits Ig-alpha and Ig-beta, associated to B cell Ag receptor, targeted Ags either to newly synthesized or to preexisting pools of MHC class II molecules, respectively. To further characterize these two Ag presentation pathways, we altered the intra-Golgi transport of newly synthesized MHC class II by stably overexpressing, in B cells, mutants of a small G protein involved in the intra-Golgi transport, Rab6. Overexpression of GTP-bound rab6 (Q72L) mutant proteins reduced the cell surface arrival of MHC class II molecules and consequently slowed down Ag presentation dependent upon newly synthesized class II molecules. In contrast, this mutant had no effect on Ag presentation dependent upon preexisting pools of class II molecules, and the overexpression of an inactive GDP-bound form of rab6 (T27N) did not affect any Ag presentation pathway. MHC class II-restricted Ag presentation pathways can therefore be distinguished by their sensitivity to the overexpression of proteins modifying the intracellular transport of newly synthesized class II molecules.
Derivation of HLA-A11/Kb transgenic mice: functional CTL repertoire and recognition of human A11-restricted CTL epitopes.
Authors: Alexander J , Oseroff C , Sidney J , Wentworth P , Keogh E , Hermanson G , Chisari F V , Kubo R T , Grey H M , Sette A
Published: December 2022

Transgenic mice expressing chimeric human (alpha1 and alpha2 HLA-A11 domains) and murine (alpha3, transmembrane, and cytoplasmic H-2Kb domains) class I molecules were derived. These mice were used as a model system to study the immunogenicity of human CTL epitopes and also to examine the aspects of Ag processing differences of mice vs man. Immunization of these mice with seven known HLA-A11-restricted CTL epitopes emulsified in IFA resulted in vigorous specific CTL responses. A larger panel of 45 A11-binding peptides was used to examine the relationship between immunogenicity in the HLA-A11/Kb transgenic mice and HLA-A11 binding capacity. Twenty-one of 28 (75%) peptides with high binding affinities (50% inhibitory concentration (IC50), 2-50 nM) and 7 of 13 (54%) intermediate binding peptides (IC50, 50-500 nM range) were immunogenic. In parallel, 19 of these peptides were used for in vitro primary immunizations of PBMC derived from HLA-A11 healthy human donors. It was found that 8 of 8 peptides that were able to elicit CTL in primary human in vitro cultures were also immunogenic in HLA-A11/Kb mice. Finally, HLA-A11/Kb transgenic mice were found to generate an A11/Kb restricted CTL response following immunization with influenza virus A/PR/8/34, suggesting that, at least to some extent, A11 epitopes are generated by transgenic mice as a result of natural in vivo processing and presentation.
CD4+ CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway.
Authors: Lesage S , Steff A M , Philippoussis F , Pagé M , Trop S , Mateo V , Hugo P
Published: December 2022

Ligation of the protein tyrosine phosphatase CD45 on both mature and immature T cells modulates the amplitude of TCR-mediated signals. In this work, we have evaluated the consequences of CD45 ligation on immature T cells, in the absence of TCR engagement. Cross-linking of CD45 on thymocytes by mAbs led to the induction of cellular death, characterized by a reduction in mitochondrial membrane potential (delta psi(m)), production of reactive oxygen species, loss in membrane asymmetry, exposure of phosphatidylserine residues, and incorporation of vital dyes. In sharp contrast to most stimuli causing thymocyte death, CD45 cross-linking did not lead to DNA degradation. Cell death was not blocked by Bcl-2 overexpression or treatment with caspase inhibitor. However, death was inhibited by the addition of scavengers of reactive oxygen species. We also established that susceptibility to CD45-mediated death is acquired during the transition of early CD4- CD8- TCR- T cell precursors into CD4+ CD8+ TCR- thymocytes and is increased with further acquisition of surface TCR on these cells. Moreover, mature thymocytes were much less sensitive to CD45 cross-linking than CD4+ CD8+ cells. We propose that during T cell development, CD45 ligation could induce the death of those immature thymocytes that do not fulfill the requirements for positive selection.
Bcl-x protects primary B cells against Fas-mediated apoptosis.
Authors: Schneider T J , Grillot D , Foote L C , Núñez G E , Rothstein T L
Published: December 2022

Primary murine splenic B cells are rendered sensitive or resistant to Fas-mediated apoptosis in a receptor-specific fashion. B cells stimulated though CD40 are Fas sensitive unless they also receive a signal though surface Ig that produces a state of resistance to Fas killing. Protection from Fas-mediated apoptosis takes time to develop and requires ongoing macromolecular synthesis; therefore, it appears to involve the induction and accumulation of one or more gene products. The role of Bcl-x was evaluated by examining the expression and function of this gene in primary B cells. bcl-x mRNA was induced by anti-IgM treatment of otherwise sensitive (CD40 ligand-treated) B cells. Bcl-x protein expression was induced by anti-IgM and appeared in a time frame that correlates well with the onset of anti-IgM-induced Fas resistance. Further, B cells from Bcl-x Tg mice were found to be resistant to Fas-mediated apoptosis. These results strongly suggest that the protection against Fas killing afforded by cross-linking surface Ig is mediated, at least in part, by an increase in Bcl-x.
Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production.
Authors: Zhang H , Peterson J W , Niesel D W , Klimpel G R
Published: December 2022

Septic shock is a major cause of death in the world. Although much is known about the role of LPS in septic shock, little is known about the role of other bacterial components. Lipoprotein (LP) is a major component of bacteria in the family Enterobacteriaceae. LP purified from Escherichia coli was shown to induce TNF-alpha and IL-6 production in peritoneal exudate macrophages obtained from LPS-responsive (C3H/HeOuJ) and LPS-nonresponsive (C3H/HeJ) mice. LP and LPS acted synergistically to induce cytokine production not only in C3H/HeOuJ macrophages but also in C3H/HeJ macrophages. These results suggest that LPS can induce cellular signaling in C3H/HeJ macrophages, and that LPS and LP activate macrophages via different receptors and/or signaling pathways. The role LP plays in septic shock was investigated using the mouse D-galactosamine model. LP induced lethal shock and in vivo production of TNF-alpha and IL-6 in both LPS-responsive and LPS-nonresponsive mice. LPS failed to induce lethal shock or in vivo cytokine production in C3H/HeJ mice. However, LP and LPS acted synergistically in inducing lethal shock and in vivo cytokine production in both LPS-responsive and LPS-nonresponsive mice. Finally, a heat-killed preparation of an E. coli mutant strain that lacked LP was shown to be less efficient than heat-killed wild-type E. coli at inducing lethal shock in C3H/HeJ mice. Collectively, these results suggest that LP and LPS induce cytokine production via different mechanisms and that LP plays an important role in septic shock induced by bacteria in the family Enterobacteriaceae.
Characterization of CD40 signaling determinants regulating nuclear factor-kappa B activation in B lymphocytes.
Authors: Hsing Y , Hostager B S , Bishop G A
Published: December 2022

CD40 signaling to B cells is important for generating an effective humoral immune response. CD40 ligation leads to B cell activation events such as proliferation, Ig secretion, isotype switching, and up-regulation of cell surface molecules, as well as the generation of memory B cells. Many of these events are dependent upon the ability of CD40 to activate the transcription factor NF-kappa B (NF-kappa B). To define the CD40 signaling components upstream of NF-kappa B activation and the functional consequences downstream of NF-kappa B activation, we examined mouse B cell transfectants expressing wild-type or mutant human CD40. Analysis of CD40 cytoplasmic domain truncation and point mutants defined a 10-amino acid CD40 cytoplasmic signaling determinant required for NF-kappa B activation. A threonine residue at position 234, previously shown to be important for CD40 association with TNF receptor-associated factor 2 (TRAF2), TRAF3, and TRAF5, was not required for NF-kappa B activation. This suggests that in B cells, CD40-induced NF-kappa B activation can occur independently of TRAF2 and TRAF5 association. NF-kappa B activation was independent of the transmembrane domain of CD40, suggesting that it is independent of p23, a molecule that associates with CD40 in a region other than the cytoplasmic domain. Proteasome-dependent inhibitory kappa B alpha (I kappa B alpha) and I kappa B beta degradation occurred downstream of CD40 ligation and preceded CD40-mediated NF-kappa B nuclear translocation. CD40- or pervanadate-mediated I kappa B tyrosine phosphorylation was not detected. NF-kappa B activation correlated with the ability of CD40 to induce Ab secretion and the up-regulation of ICAM-1 and LFA-1. However, NF-kappa B activation was insufficient for CD40-mediated up-regulation of B7-1, Fas, and CD23.