bluuebunny/crossref_metadata_2025 huggingface dataset:
Accelerated collagen-induced arthritis in IFN-gamma receptor-deficient mice.
Authors: Vermeire K , Heremans H , Vandeputte M , Huang S , Billiau A , Matthys P
Published: December 2022

Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis. Here, we describe experiments showing that IFN-gamma receptor knockout (IFN-gammaR alpha KO) mice of the DBA/1 strain develop CIA more readily than their wild-type counterparts. Symptoms of disease started 10 days earlier and the cumulative incidence of arthritis was significantly higher in the mutant mice than in wild-type mice. Similarly, accelerated onset of the disease was also found in wild-type DBA/1 mice treated with neutralizing mAbs against IFN-gamma. Histologic examination of the joints revealed a massive infiltration of the synovium with mononuclear cells and neutrophils, hyperplasia, and severe pannus formation in IFN-gammaR alpha KO mice when such inflammatory lesions were not yet detectable in wild-type mice. Serum levels of anti-collagen type II Abs, including total IgG and IgM, as well as IgG1, IgG2a, and IgG2b isotypes were found to be lower in the mutant mice. IL-2 and IL-4 remained undetectable in sera of both groups of mice, but did appear in the circulation after anti-CD3 Ab challenge. Significantly higher IL-2 and lower IL-4 serum levels were found in anti-CD3-challenged IFN-gammaR alpha KO mice than in wild-type counterparts, both at an early and at a later stage of the disease. These observations indicate that endogenous IFN-gamma counteracts development of collagen-induced arthritis and suggest that IFN-gamma does so by up-regulating IL-4 production and/or down-regulating IL-2 production. The data are in line with the concept of a pathogenic role of Th1-type cellular immunity in CIA in spite of a decreased Ab response to collagen type II.
Macrophage migration inhibitory factor is involved in the pathogenesis of collagen type II-induced arthritis in mice.
Authors: Mikulowska A , Metz C N , Bucala R , Holmdahl R
Published: December 2022

To determine the importance of macrophage migration inhibitory factor (MIF) in the development of arthritis we used an experimental model for rheumatoid arthritis, collagen type II (CII)-induced arthritis in mice. Treatment with neutralizing anti-MIF Abs before immunization of (B10.Q x DBA/1)F1 with CII led to delayed onset and lowered frequency of arthritis. This was associated with lower levels of IgG2a to CII in MIF-depleted mice. The proliferative response to CII was stronger in the anti-MIF-treated mice, whereas no significant effects were seen on Ag-induced IFN-gamma production in response to CII or on the total serum Ab levels in response to CII. These results provide the first experimental evidence of a role for MIF in the pathogenesis of autoimmune disease.
Enhancement of gp130-mediated tyrosine phosphorylation of STAT3 and its DNA-binding activity in dexamethasone-treated AIDS-associated Kaposi's sarcoma cells: selective synergy between dexamethasone and gp130-related growth factors in Kaposi's sarcoma cell proliferation.
Authors: Murakami-Mori K , Mori S , Taga T , Kishimoto T , Nakamura S
Published: December 2022

The soluble IL-6R (slL-6R alpha)/IL-6 complex and oncostatin M (OM), which exert biologic activities through the signal-transducing protein gp130, are potent growth factors for AIDS-associated Kaposi's sarcoma (KS) cells. Clinical observations indicate that glucocorticoid therapy is a possible risk factor in KS; however, little is known of specific interactions in KS cells between glucocorticoid and gp130-related growth factors. We obtained evidence that dexamethasone (Dex), in a synergistic manner, enhances gp130-mediated growth of KS cells. Anti-gp130 Abs or the glucocorticoid antagonist RU-486 abolished this synergistic effect. In addition, Dex had additive but not synergistic effects on stimulation of KS cell growth with IL-1beta or TNF-alpha, the signals of which are not mediated through gp130. Immunoblot analysis revealed sIL-6R alpha/IL-6- or OM-induced tyrosine phosphorylation of a similar set of proteins in KS cells, and which was augmented significantly in Dex-treated KS cells. Stimulation of KS cells with sIL-6R alpha/IL-6 or OM induced rapid tyrosine phosphorylation of the transcription factor STAT3, and Dex significantly enhanced the accumulation of tyrosine-phosphorylated STAT3. Electrophoretic mobility shift assays showed sIL-6R alpha/IL-6- or OM-induced DNA-binding activity of STAT3 in KS cells, and Dex further increased this activity. Thus, Dex appears to participate in the gp130-STAT3 signaling and transcriptional events by enhancing STAT3 activation, thereby leading to selective synergistic stimulation of KS cell growth with Dex and the gp130-related growth factors.
Expression of IFN-inducible protein-10 in chronic hepatitis.
Authors: Narumi S , Tominaga Y , Tamaru M , Shimai S , Okumura H , Nishioji K , Itoh Y , Okanoue T
Published: December 2022

Chemokines such as IFN-inducible protein-10 (IP-10) and JE/monocyte chemotactic protein-1 (MCP-1) are induced in the murine liver in a tissue-specific manner. We examined whether IP-10 and MCP-1 are pathologically involved in chronic hepatitis. Whereas the serum levels of IP-10 and MCP-1 in patients with chronic persistent hepatitis C were elevated compared with those in normal volunteers, both chemokine levels were further significantly higher in patients with the active form (chronic active hepatitis (CAH)). The elevated IP-10 level was not a general phenomenon of inflammation, because it was not seen in patients with rheumatoid arthritis, whereas MCP-1 levels were elevated to the same extent in both patient groups. Better responsiveness to IFN therapy in CAH was related to lesser grades of necroinflammatory activity and was predicted by the lower IP-10 and higher MCP-1 levels. IP-10 levels in patients cured by IFN therapy decreased to the levels in normal volunteers, while the MCP-1 levels only slightly decreased. Serum levels of both chemokines in patients who were not cured remained unchanged after IFN therapy. In situ hybridization analysis of CAH revealed that IP-10 mRNA was expressed mainly in hepatocytes around intralobular focal and periportal piecemeal necrosis, while some MCP-1 mRNA was expressed in some sinusoidal cells. These results suggested that IP-10 plays a specific role in the intralobular accumulation of mononuclear cells and/or the death of hepatocytes in chronic hepatitis.
Specialization of mucosal follicular dendritic cells revealed by mucosal addressin-cell adhesion molecule-1 display.
Authors: Szabo M C , Butcher E C , McEvoy L M
Published: December 2022

Follicular dendritic cells (FDC) in the mucosal lymphoid organs, Peyer's patches (PP), display mucosal vascular addressin-cell adhesion molecule-1 (MAdCAM-1). MAdCAM-1 decorates interlacing dendritic cells throughout PP follicles and is accentuated on FDC of the germinal center (GC) light zone and on "junctional" dendritic cells overlapping the B/T border and subepithelial dome region, sites associated with microenvironmental homing decisions. In contrast, MAdCAM-1 is rarely displayed by coronal or junctional FDC in peripheral lymph node follicles and is largely confined to the GC after lymph node immunization. FDC-associated MAdCAM-1 plays a prominent role in lymphocyte adhesion to GC in PP frozen sections and participates significantly in binding to GC in chronically stimulated lymph nodes and spleen, but not to GC in lymph nodes after primary immunization (where binding is dominated by vascular cell adhesion molecule-1). Differential display of MAdCAM-1 by FDC could contribute to the specialization of mucosal vs nonmucosal immune responses.
Close phenotypic and functional similarities between human and murine alphabeta T cells expressing invariant TCR alpha-chains.
Authors: Davodeau F , Peyrat M A , Necker A , Dominici R , Blanchard F , Leget C , Gaschet J , Costa P , Jacques Y , Godard A , Vie H , Poggi A , Romagné F , Bonneville M
Published: December 2022

Several studies have demonstrated the existence of a murine NK1.1+ alphabeta T cell subset expressing V alpha14+ TCR alpha-chains with highly conserved invariant junctional sequences and able to secrete Th2 cytokines when exposed to CD1+ stimulator cells. In humans, alphabeta T cells carrying invariant V alpha24+ TCR alpha-chains highly homologous to those expressed by murine NK1.1 cells have been recently described. Here we show that these cells (referred to as V alpha24inv T cells) and murine NK1.1+ alphabeta T cells resemble each other in several ways. First, like their murine counterparts, T cells expressing high levels of V alpha24inv TCRs can be either CD4- CD8- double negative (DN) or CD4+, but they never express heterodimeric CD8 molecules. Second, most V alpha24inv T cells are brightly stained by NKRP1-specific mAb but not by mAb directed against other type II transmembrane proteins of the NK complex. Third, DN and particularly CD4+ V alpha24inv T cells are greatly enriched for IL-4 producers. The concomitant expression of highly conserved TCRs of a particular set of NK markers and of Th2 cytokines in human and murine alphabeta T cells suggests a coordinate acquisition of these phenotypic and functional properties. Furthermore, the relatively high frequency of human V alpha24inv T cells, which are presently shown to represent on average 1/500 PBL, and the high interindividual variations of the size of this cell subset under physiologic conditions go for a major role played by alphabeta T cells carrying invariant TCR in a large array of immune responses.
An octamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility during myelomonocytic differentiation.
Authors: López-Rodríguez C , Zubiaur M , Sancho J , Concha A , Corbi A L
Published: December 2022

The integrin CD11c/CD18 mediates leukocyte adhesion to endothelium and other cell types and is a receptor for LPS, iC3b, and fibrinogen. CD11c expression is restricted to myeloid and activated B cells, is regulated during leukocyte differentiation, and constitutes a diagnostic tool for hairy cell leukemia. Mapping of in vivo DNA-protein interactions in the CD11c proximal promoter revealed three adjacent myeloid-specific interactions, one of which lies on an octamer consensus sequence, ATTT GCAT (Oct185). Oct185 disruption increased the CD11c promoter activity while decreasing its myeloid differentiation responsiveness, indicating that Oct185 contributes to the activity of the CD11c promoter and suggesting that Oct185 is a negative regulatory element whose function changes during myeloid differentiation. Oct185 is recognized by the ubiquitous Oct-1 factor in all cell lineages and by Oct-2 in B lymphoid lineage cells. Unexpectedly, Oct-2 binding to Oct185 was induced de novo upon monocytic differentiation of U937 and HL-60 cells but not during HL-60 granulocytic differentiation, as determined by electrophoretic mobility shift assays and immunochemical studies, and Oct-2 complexes were also observed in cultured adherent monocytes. Western blotting showed that the pattern of Oct-2 isoforms in myeloid cells is similar to that seen in B cells. The Oct-2 up-regulated expression in differentiating myeloid cells and its binding to the Oct185 negative regulatory element suggests its involvement in the differentiation-regulated activity of the CD11c promoter and might represent an important parameter for the myeloid- and B cell-restricted expression of the CD11c/CD18 integrin and other molecules with similar patterns of expression.
Allotype-associated variation in the human gamma3 switch region as a basis for differences in IgG3 production.
Authors: Pan Q , Rabbani H , Mills F C , Severinson E , Hammarström L
Published: December 2022

High and low serum concentrations of IgG3 are associated with the human G3 m(b) and G3 m(g) allotypes, respectively. We previously hypothesized that a low frequency of switching is the most likely defect in (g) allotype-positive individuals, and therefore analyzed the structure, recombination breakpoints, and binding of nuclear proteins to the switch (S)gamma3 regions of these two allotypes. There are no allotype-associated differences in the length and basic structure of the Sgamma3, since both contain eighteen 79-bp repeats. However, we found a number of allotype-associated nucleotide changes. As in the mouse system, there is a preferential switching to the B site, or switch nuclear protein/nuclear factor-kappaB motif, with a clustering of switch breakpoints at the most 5' residue of the B site. The B site sequence used most frequently in switching was found to be mutated at this nucleotide in the (g) allotype-associated Sgamma3. This change was shown by electrophoretic mobility shift assay to alter the binding of the switch nuclear protein/nuclear factor-kappaB protein to the B site. Taken together, these data suggest that polymorphism within Sgamma3 may contribute to allotype-associated differences in IgG3 switching, and that specific sequences within the Sgamma3 79-bp repeats could be mechanistically important for switch recombination.
Four conserved promoter motifs regulate transcription of the gene encoding human complement component C2.
Authors: Sullivan K E
Published: December 2022

The early complement components of the classical activation pathway of the complement cascade include the components C1, C4, C2, and C3. These components act in concert to opsonize bacteria, clear immune complexes, and produce inflammatory mediators. They are not structurally homologous nor are they coordinately regulated. The expression of the early complement components is divergent in terms of cytokine responsiveness and tissue specificity. The only pattern of expression shared by the early complement components is inducibility by gamma-IFN and expression in cells of hepatic or monocytic lineage. Nevertheless, four novel conserved promoter motifs were identified in the 5' flanking region of multiple early complement component promoters. Mutation of these four motifs in the C2 promoter decreased transcription in hepatoma cells in transient transfection analyses, and a synthetic promoter consisting of just the four motifs supported transcription in hepatoma cells. Electrophoretic mobility shift assays demonstrate that three of the four conserved elements bind DNA-binding proteins in a tissue-specific manner. One DNA-binding protein is expressed ubiquitously, but the other three are restricted to cells of monocytic or hepatic lineage. Two of the DNA-binding proteins appear to be members of the zinc-finger family of transcription factors. Therefore, these four motifs appear to bind DNA-binding proteins that may function in the tissue-specific expression of C2. Conservation of these four motifs in multiple early complement component genes suggests that these may represent a conserved transcriptional strategy.
The transcription factor B cell-specific activator protein (BSAP) enhances both IL-4- and CD40-mediated activation of the human epsilon germline promoter.
Authors: Thienes C P , De Monte L , Monticelli S , Busslinger M , Gould H J , Vercelli D
Published: December 2022

Induction of isotype switching to a particular C(H) gene correlates with the transcriptional activation of the same gene in germline configuration. Induction of correctly spliced germline transcripts is necessary to target a switch region for recombination and switching. Different cytokines activate transcription at different germline promoters. Because binding sites for the B cell-specific transcription factor BSAP are located upstream of several switch regions in the Ig locus, BSAP might play a role in isotype switching by regulating germline transcription. We investigated whether BSAP plays a role in the transcriptional regulation of the epsilon germline promoter in human B cells. We identified human EBV-negative B cell lines that express epsilon germline transcripts upon stimulation with IL-4. Electrophoretic mobility shift assay analysis showed that the human epsilon germline promoter binds BSAP. BSAP activity was expressed constitutively and was not affected by stimulation with IL-4 and/or anti-CD40 mAb. Reporter assays with constructs containing a luciferase gene driven by the epsilon germline promoter, with or without mutations in the BSAP binding site, showed that BSAP plays a role in both IL-4-dependent induction and CD40-mediated up-regulation of human epsilon germline transcription. Furthermore, epsilon germline promoter activity was abrogated in REH cells that express a BSAP polypeptide truncated in the trans-activation domain. Among the transcription factors that regulate epsilon germline expression, BSAP is unique, in that it is B cell-specific and is at the merging point of two signaling pathways that are distinct but both critical for the induction of IgE switching.
Transcriptional activation of vascular cell adhesion molecule-1 gene in vivo and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock.
Authors: Essani N A , Bajt M L , Farhood A , Vonderfecht S L , Jaeschke H
Published: December 2022

Polymorphonuclear leukocytes (neutrophils) can cause hepatic parenchymal cell injury during endotoxin (ET) shock. Because adhesion molecules are critical for inflammatory cell damage, the role of vascular cell adhesion molecule-1 (VCAM-1) was studied in the pathophysiology of ET shock. ET-sensitive mice (C3Heb/FeJ) were treated with 700 mg/kg galactosamine in combination with 100 microg/kg Salmonella abortus equi ET, 15 microg/kg TNF-alpha, or 13 to 23 microg/kg IL-1. VCAM-1 mRNA formation was strongly activated in animals treated with ET, TNF-alpha, or IL-1. In contrast, only TNF-alpha and IL-1, not ET, induced VCAM-1 gene transcription in livers of ET-resistant mice (C3H/HeJ). Immunohistochemistry and isolation of liver cells during endotoxemia indicated that VCAM-1 mRNA and protein were only formed in endothelial cells and Kupffer cells, not in hepatocytes. Galactosamine/ET induced neutrophil accumulation in sinusoids (515 +/- 30 neutrophils/50 high power fields) followed by transmigration at 7 h. At that time, severe liver injury was observed (necrosis, 53 +/- 5%). An anti-VCAM-1 Ab (3 mg/kg) attenuated the area of necrosis by 60%. The Ab reduced neutrophil transmigration by 84%, but had no effect on the total number of cells in the liver vasculature. Flow cytometric analysis identified the presence of very late Ag-4 on mouse peripheral neutrophils. Our data demonstrated cytokine-dependent VCAM-1 gene transcription and protein expression in the liver during endotoxemia. Neutrophils were able to use very late Ag-4/VCAM-1 interactions to transmigrate into liver parenchyma in vivo. Preventing transmigration by blocking VCAM-1 protected hepatocytes against neutrophil-induced injury.
TGF-beta attenuates the class II transactivator and reveals an accessory pathway of IFN-gamma action.
Authors: Nandan D , Reiner N E
Published: December 2022

In the present report, the induction of the HLA-DRA gene in response to IFN-gamma is shown to be selectively attenuated by TGF-beta. Thus, the accumulation in response to IFN-gamma of mRNA for the DRA gene, but not for the guanylate binding protein-2 gene, is markedly reduced in the presence of TGF-beta. Moreover, the data presented show that the mechanism by which TGF-beta inhibits expression of DRA involves attenuation of the class II transactivator (CIITA) gene. This conclusion is based on the finding that induction of CIITA gene expression in response to IFN-gamma is completely abrogated in TGF-beta-treated cells. In contrast, TGF-beta did not affect IFN-gamma-induced tyrosine phosphorylation of Jak1, Jak2, or the signal transducer and activator of transcription-1 (Stat1). TGF-beta also did not inhibit the appearance of IFN-gamma-activated, Stat1 DNA-binding activity in intact cells. Thus, the effects of TGF-beta on CIITA could not be explained by altered signaling through Jak-Stat1. Potential alternative targets for the inhibitory effects of TGF-beta were identified in renaturation tyrosine kinase assays, which revealed three IFN-gamma-activated protein tyrosine kinases that, in contrast to the Janus kinases, are sensitive to TGF-beta. These findings 1) indicate that inhibition of MHC class II gene expression by TGF-beta involves attenuation of the CIITA gene independently of effects on Janus kinases, 2) provide direct evidence that IFN-gamma activates both Janus and non-Janus protein tyrosine kinases, and 3) identify an accessory pathway of IFN-gamma action involving tyrosine kinases that, unlike the Jak-Stat1 pathway, are impaired by TGF-beta.
Differential interaction of nuclear factors with the PRE-I enhancer element of the human IL-4 promoter in different T cell subsets.
Authors: Li-Weber M , Salgame P , Hu C , Davydov I V , Krammer P H
Published: December 2022

The immunomodulatory cytokine IL-4 affects cells of most hemopoietic lineages. IL-4 is secreted by activated Th2 but not Th1 cells and plays a major role in the immune response by modulating the differentiation of naive Th cells toward the Th2 phenotype. We have previously identified an enhancer element, PRE-I, that is essential for the function of the human IL-4 promoter. To investigate the mechanisms responsible for tissue-specific expression of the IL-4 gene, we analyzed nuclear factors binding to the PRE-I site and compared the binding activities of these factors to the IL-4 promoter of Th1 and Th2 cells. We show that PRE-I interacts with PMA- and PMA/ionomycin-inducible, cyclosporin A-sensitive nuclear factors. Using anti-C/EBPbeta (NF-IL6), anti-C/EBPdelta (NF-IL6beta), anti-NF-ATc, anti-NF-ATp, anti-Fos, and anti-Jun Abs we demonstrate that the previously identified PRE-I binding factor POS-1 is composed of different transcription factors in different Th cell subsets. In the IL-4-producing Th0-like human Jurkat and mouse EL-4 cells, POS-1 (designated POS-1a) contains NF-IL6beta and Jun. In the mouse Th2 D10 cells and in the human Th2 clones, POS-1 (designated POS-1b) contains NF-IL6beta, Jun, and NF-ATc/p. In contrast, POS-1 was not found in nuclear extracts of human Th1 clones. These findings suggest that PRE-I may play a role in the differential regulation of IL-4 gene expression levels.
Control of Leishmania major infection in BALB/c mice by inhibition of early lymphocyte entry into peripheral lymph nodes.
Authors: Laskay T , Wittmann I , Diefenbach A , Röllinghoff M , Solbach W
Published: December 2022

A single i.p. injection with the anti-CD62L (anti-L-selectin) mAb Mel-14 before parasite challenge protected BALB/c mice from the otherwise lethal infection with Leishmania major. The Mel-14 mAb treatment resulted in a significant (>90%) decrease in cellularity of the popliteal lymph node (PLN) with a decrease in the proportion of CD4+ cells and an increase of the proportion of B220+ cells. Furthermore, both activated cells (CD25+ and CD69+) and cells of the memory phenotype (CD45RBdull CD44high) were significantly enriched in PLN from Mel-14-treated BALB/c mice. After infection with L. major, the otherwise massive cellular infiltration in the draining PLN was completely blocked in the Mel-14-treated mice, and in these animals the high representation of both activated and memory cells in PLN remained characteristic for the first days of infection. The protective effect was found to be associated with a markedly increased production of IFN-gamma and with a decrease in IL-4 production upon restimulation of PLN and spleen cells with L. major Ag in vitro. The cured mice were found to be resistant against a secondary challenge with the parasites. These data suggest that the induction of a nonprotective Th2 response to L. major is associated with the entry of lymphocytes from the recirculating pool into the draining LN. The Mel-14-induced changes in the lymphoid microenvironment of the draining peripheral LN appear to favor the development of a protective Th1 cell-mediated immune response against the parasite.
Selective inhibition of human and mouse natural killer tumor recognition using retroviral antisense in primary natural killer cells: involvement with MHC class I killer cell inhibitory receptors.
Authors: Ortaldo J R , Mason A T , Mason L H , Winkler-Pickett R T , Gosselin P , Anderson S K
Published: December 2022

The natural killer tumor recognition (NK-TR) protein has been shown to be a necessary component for the killing of NK-sensitive and virus-infected targets by the rat RNK-16 cell line. Class I-recognizing killer cell inhibitory receptors (KIR) have been found in the human (p58; NKAT family) and mouse (Ly-49 family). The principal functional characteristic of these receptors is their ability to block NK cell lysis by recognition of selected class I molecules on target cells. In the present study, we examined whether abrogation of NK-TR expression by retroviral infection of primary human or mouse NK cells with virus-producing antisense NK-TR also would demonstrate loss of non-MHC-restricted killing and whether the NK-TR was associated with KIR function in humans or with Ly-49 in the mouse. Using short term culture of fresh human or mouse NK cells, antisense NK-TR-treated NK cells demonstrated strong selective reduction of NK cytotoxicity. NK-TR was necessary for lytic activity even when KIR function was blocked by Ab in experiments involving NK3.3 lysis of HLA.cw3-expressing targets or killing of Dd targets by Ly-49A+ or Ly-49G2+ mouse NK cells. These studies extend our previous studies in rat NK cell lines to demonstrate that primary mouse and human NK cells require NK-TR for non-MHC-restricted lysis of tumor and virus-infected targets. In addition, the reversal of KIR or Ly-49 inhibition of NK cell lysis requires NK-TR expression for cellular killing in both human and mouse.
Proinflammatory functions of IL-2 in herpes simplex virus corneal infection.
Authors: Tang Q , Chen W , Hendricks R L
Published: December 2022

Herpes simplex virus type 1 infection of corneas can lead to blinding inflammation in the corneal stroma, which is referred to clinically as herpes stromal keratitis. In our mouse model of this prevalent human disease, a heavy polymorphonuclear neutrophil (PMN) infiltration of the infected cornea leads to progressive tissue destruction. This inflammatory process can be abrogated by in vivo depletion of CD4 T lymphocytes and by neutralization of the cytokines IL-2 and IFN-gamma. The goal of this study was to define the mechanisms by which IL-2 mediates the corneal inflammation. Systemic neutralization of IL-2 after the onset of corneal disease resulted in a rapid regression of inflammation and complete resolution in 50% of the treated mice. The disease remission was associated with loss of IFN-gamma expression in the cornea, as determined by immunohistochemistry, and a significant reduction of IFN-gamma mRNA, as measured by a semiquantitative reverse transcription-PCR analysis. Within 48 h after anti-IL-2 mAb administration, the PMN chemotactic gradient in the infected corneas was abolished, and those PMN that were already present in the central cornea exhibited clear signs of apoptotic cell death. Our results demonstrate that IL-2 mediates corneal inflammation by 1) regulating local IFN-gamma production in an autocrine or a paracrine fashion, 2) establishing a PMN chemotactic gradient, and 3) maintaining PMN viability in the cornea. These results suggest that IL-2 might be targeted for therapeutic intervention in this blinding disease.
Lymphotactin gene expression in mast cells following Fc(epsilon) receptor I aggregation: modulation by TGF-beta, IL-4, dexamethasone, and cyclosporin A.
Authors: Rumsaeng V , Vliagoftis H , Oh C K , Metcalfe D D
Published: December 2022

Recruitment of lymphocytes is a prominent feature of allergic inflammation. However, the mechanisms by which lymphocytes are attracted to such sites are not understood. Recently, cDNAs encoding a lymphocyte-specific chemokine, lymphotactin (Ltn), were isolated from mouse pro-T cell and human CD8+ T cell libraries, leading us to hypothesize that mast cells might also produce Ltn. Using the reverse transcriptase-PCR and Northern blot analysis, we found that the Ltn gene is inducible in C1.MC/C57.1 and murine bone marrow-cultured mast cells (BMCMC) by Fc(epsilon)RI aggregation. Activation of a human mast cell (HMC-1) or basophil cell line (KU812) similarly led to transcription of Ltn. Fc(epsilon)RI aggregation-dependent Ltn mRNA expression was detected by 1 to 2 h, maximal at 6 h, independent of de novo protein synthesis, and was inhibited by cyclosporin A and dexamethasone. Compared with macrophage inflammatory protein alpha (MIP-1alpha), Fc(epsilon)RI-dependent Ltn and MIP-1alpha mRNA levels were up-regulated by IL-4, but not IFN-gamma, although higher levels of IL-4 (100 and 1000 U/ml) inhibited Ltn expression only; and TGF-beta preferentially enhanced Fc(epsilon)RI-dependent Ltn mRNA levels, suggesting that Ltn and MIP-1alpha have shared and unique regulatory mechanisms. A rabbit polyclonal Ab against a synthetic peptide was developed for use in immunoblot analysis and detected a 15-kDa Ltn protein within mast cell pellets and in the supernatants of mast cells following Fc(epsilon)RI aggregation. Ltn is thus expressed in mast cells and may contribute to the recruitment of lymphocytes to areas of allergic inflammation.
Molecular adjuvant effects of a conformationally biased agonist of human C5a anaphylatoxin.
Authors: Tempero R M , Hollingsworth M A , Burdick M D , Finch A M , Taylor S M , Vogen S M , Morgan E L , Sanderson S D
Published: December 2022

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating Ab responses against peptide epitopes derived from human MUC1 glycoprotein and the human mu and kappa opioid receptors. C57BL6 mice were immunized with the MUC1 epitope (YKQGGFLGL); the C5a agonist (YSFKPMPLaR); YSFKPMPLaR and YKQGGFLGL together, but unconjugated; a C5a-active, MUC1 epitope construct (YKQGGFLGLYSFKPMPLaR); and a C5a-inactive, reversed moiety construct (YSFKPMPLaRYKQGGFLGL). High Ab titers specific for the MUC1 epitope were observed only in mice immunized with the C5a-active epitope construct. Similar results were obtained in BALB/c mice immunized with the C5a-active, MUC1 epitope construct. Abs from the sera of the C57BL6 mice were predominately of the IgG2a, IgG2b, and IgM isotypes and were reactive against human recombinant MUC1 and MUC1 expressed by the Panc-1 M1F.15 pancreatic cell line. When compared with the corresponding KLH-epitope conjugates in C57BL6 mice, the epitope-C5a agonist constructs produced titers of specific IgG Abs of isotypes distinct from those generated by the keyhole limpet hemocyanin-epitope conjugates. Rabbits immunized with a mu opioid receptor epitope-C5a agonist construct (GDLSDPCGNRTNLGGRDSLYSFKPMPLaR) or a kappa opioid receptor epitope-C5a agonist construct (FPGWAEPDSNGSEDAQLYSFKPMPLaR) generated high titer, epitope-specific Ab responses. Ab titers generated in response to the opioid epitope-C5a agonist constructs were comparable to those generated by the opioid KLH-epitope conjugates. The results of this study are discussed in terms of possible mechanisms by which the conformationally biased C5a agonist serves as a molecular adjuvant.
Evidence for the role of an altered redox state in hyporesponsiveness of synovial T cells in rheumatoid arthritis.
Authors: Maurice M M , Nakamura H , van der Voort E A , van Vliet A I , Staal F J , Tak P P , Breedveld F C , Verweij C L
Published: December 2022

In rheumatoid arthritis (RA), T cells isolated from the synovial fluid (SF) show impaired responses to mitogenic stimulation compared with T cells from the peripheral blood (PB). Here it is reported that hyporesponsiveness of SF T cells correlated with a significant decrease in the levels of the intracellular redox-regulating agent glutathione (GSH). GSH was decreased in both CD4+ (p = 0.0022) and CD8+ (p = 0.0010) SF T cell subsets compared with PB CD4+ and CD8+ T cells in RA patients. Levels of thioredoxin (TRX), another key redox mediator, previously found to be secreted under conditions of oxidative stress, were found to be significantly increased in SF compared with plasma samples of RA patients (p = 0.005). Increased levels of TRX in the SF of inflamed joints was found to be associated with RA when compared with other arthritides (p = 0.007). Restoration of GSH levels in SF T cells with N-acetyl-L-cysteine (NAC), enhanced mitogenic induced proliferative responses and IL-2 production. Collectively, these data impute an important role to an altered redox state in the hyporesponsiveness of joint T cells in patients with RA.
Clonal expansion within the CD4+CD57+ and CD8+CD57+ T cell subsets in chronic lymphocytic leukemia.
Authors: Serrano D , Monteiro J , Allen S L , Kolitz J , Schulman P , Lichtman S M , Buchbinder A , Vinciguerra V P , Chiorazzi N , Gregersen P K
Published: December 2022

The role of T cells in chronic lymphocytic leukemia (CLL) has not been extensively investigated, since the most prominent cellular abnormality in CLL involves the clonal expansion of B cells. In this study we have undertaken a comprehensive analysis of the CD4+ and CD8+ T cell repertoire in a population of CLL patients (n = 19) and age-matched controls (n = 22). The TCR repertoire analysis was performed using a multiplex PCR assay for CDR3 length, an approach that allows for the detection of underlying oligoclonality in complex T cell populations. We established that oligoclonality was substantially more frequent in both the CD4+ and CD8+ T cell populations of CLL patients than in the age-matched controls (p < 0.001). Using three-color FACS analysis with a panel of TCRV segment-specific mAbs, we also established that oligoclonal expansions are predominantly found in the CD57+ subset of both the CD4+ and CD8+ T cell populations. The frequency of the CD57 marker on CD4+ T cells was increased in the setting of CLL (% CD57 = 14.8 +/- 13.0%) compared with that in normal controls (% CD57 = 3.3 +/- 3.0%; p < 0.001). An elevated frequency of CD4+CD57+ T cells was correlated with more advanced disease. Similarly, the most extreme oligoclonal expansions of CD4+CD57+ T cells occurred in CLL patients who had progressed beyond Rai stage 0. These data document profound alterations in the T cell repertoire of CLL patients and point to a role for clonal T cell populations in the pathogenesis of this disease.
Down-regulation of surface receptors for TNF and IL-1 on circulating monocytes and granulocytes during human endotoxemia: effect of neutralization of endotoxin-induced TNF activity by infusion of a recombinant dimeric TNF receptor.
Authors: van der Poll T , Coyle S M , Kumar A , Barbosa K , Agosti J M , Lowry S F
Published: December 2022

Leukocytes rapidly lose their surface receptors for TNF and IL-1 upon exposure to various stimuli in vitro. We sought to determine by FACS analysis changes in the expression of TNF receptors (TNFR) and type II IL-1R on circulating monocytes and granulocytes during endotoxemia in vivo, and the role of endogenous TNF in these changes. Twelve healthy subjects received an i.v. injection with LPS (2 ng/kg), directly preceded by a 30-min infusion of either a recombinant human dimeric TNFR type II-IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) or vehicle (n = 6). LPS administration was associated with decreases in the expression of types I and II TNFR and type II IL-1R on both monocytes and granulocytes. Treatment with TNFR:Fc completely neutralized LPS-induced TNF activity (p < 0.0001 vs LPS only), modestly blunted the decrease in monocyte TNFR (p < 0.05), but did not influence reduced expression of granulocyte TNFR or monocyte/granulocyte type II IL-1R. In separate experiments, rTNF added to whole blood reduced cellular type I and type II TNFR expression by an effect on the type I TNFR; TNF did not (monocytes) decrease or only marginally (granulocytes) decreased type II IL-1R expression. LPS induces down-modulation of monocyte and granulocyte receptors for TNF and IL-1 in humans in vivo. TNF is involved in reduced monocyte TNFR expression during endotoxemia.
Early endosomes and a late endocytic compartment generate different peptide-class II MHC complexes via distinct processing mechanisms.
Authors: Griffin J P , Chu R , Harding C V
Published: December 2022

Class II MHC Ag-processing compartments and mechanisms were compared for four antigenic epitopes from hen egg lysozyme (HEL) and RNase. T cell assays on subcellular fractions of Ag-pulsed macrophages detected the initial appearance of HEL-(48-61):I-Ak, HEL-(34-45):I-Ak, and RNase-(90-105):I-Ek complexes in a high density late endocytic compartment. In contrast, RNase-(42-56):I-Ak complexes never appeared in high density compartments, but were rapidly generated in low density endosomes. This early endosomal processing mechanism was 1) chloroquine inhibitable; 2) less sensitive than the late endocytic mechanism to 20 degrees C inhibition; 3) partially resistant to depletion of nascent class II MHC molecules with brefeldin A, suggesting some use of recycled class II MHC molecules, whereas the late endocytic processing mechanism was blocked by brefeldin A; and 4) involved in the processing of a DM-independent complex (RNase-(42-56):I-Ak). Thus, distinct processing compartments and mechanisms are identified for different epitopes even within a single Ag.
The mechanism of in vitro T helper cell type 1 to T helper cell type 2 switching in highly polarized Leishmania major-specific T cell populations.
Authors: Mocci S , Coffman R L
Published: December 2022

We have previously demonstrated that highly polarized CD4+ Th1 cells isolated from Leishmania major-infected mice could be switched to a Th2-like phenotype when cultured for 1 wk in the presence of APC, L. major Ag, and IL-4, suggesting that the reversion of a differentiated Th response could occur at the population level. To investigate the cellular basis for this population switch, CD4+ lymph node cells from Th1-polarized L. major-infected mice were separated into two subsets based on the level of expression of L-selectin (Mel-14), and each subset was stimulated with APC and IL-2 for 1 wk in the presence or the absence of IL-4. Mel-14low T cells contained all of the initial Th1 activity and retained their Th1 phenotype when cultured with IL-4. In contrast, Mel-14high T cells did not produce cytokines upon challenge with L. major Ag, but gave rise to a Th2-like population after culture with IL-4. Thus, the newly induced Th2 population was derived from undifferentiated cells distinct from the Th1 cells present in the starting population. This undifferentiated Th precursors could be induced to develop into either Th1 or Th2 cells and were not recent thymic emigrants as they were present in mice thymectomized before infection. These experiments show that a chronically stimulated and highly polarized Th1 population consisted of both precursor T cells able to differentiate into Th2 cells and cells fully differentiated into Th1 cells that could not be induced to switch their pattern of cytokine production.
TGF-beta 1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis.
Authors: Riedl E , Strobl H , Majdic O , Knapp W
Published: December 2022

Our previous studies demonstrated that TGF-beta 1 is required for efficient in vitro generation of dendritic cells (DC) from CD34+ progenitor cells under serum-free conditions. Here we show that TGF-beta 1 promotes the growth and differentiation of DC primarily by protecting the viability of DC precursors and not by enhancing their proliferative response. Addition of TGF-beta 1 to TNF-alpha, granulocyte-macrophage CSF, and stem cell factor-supplemented cultures had no significant effect on the proportions of cycling cells. Already at 72 h of culture, however, the proportion of apoptotic cells was reduced by more than 60% in the presence of TGF-beta 1. This early protective effect of TGF-beta 1 correlates with the outgrowth of higher numbers and proportions of CD1a+ DC at day 7 of culture. It also correlates with a significantly reduced Fas/APO-1 expression on TGF-beta 1 cultured cells. In contrast, granulomonocytic cells, also arising under these culture conditions, are not affected to such an extent. They are found at equal proportions, both in the presence and absence of TGF-beta 1. The striking DC growth-promoting effect of TGF-beta 1 could only be observed when both TGF-beta 1 and TNF-alpha were present in the cultures. TGF-beta 1, in the absence of TNF-alpha, rather inhibited than enhanced cell expansion. Thus, for optimal in vitro DC development to occur, all four cytokines must obviously act in concert and the combination of TGF-beta 1 with TNF-alpha seems to be particularly critical.
Immune restoration by fetal pig thymus grafts in T cell-depleted, thymectomized mice.
Authors: Zhao Y , Fishman J A , Sergio J J , Oliveros J L , Pearson D A , Szot G L , Wilkinson R A , Arn J S , Sachs D H , Sykes M
Published: December 2022

Donor-specific tolerance can be induced across a discordant xenogeneic barrier in T/NK cell-depleted, thymectomized (ATX) B10 mice by grafting of fetal pig thymic and liver tissue (FP THY/LIV) under the kidney capsule. We have now examined the phenotype and function of murine T cells that develop in FP THY/LIV grafts in these mice. Mouse CD4+ T cells reached normal levels in PBL by 14 wk, and were maintained up to 30 wk. Similar proportions of splenic CD4+ cells expressed the naive phenotype (CD45RBhighMEL-14+CD44low) in FP THY/LIV graft recipients and euthymic control mice. These CD4 cells were functional, demonstrating normal proliferative responses and up-regulation of CD25 and CD69 after activation by mitogens or alloantigens. They proliferated in response to the protein Ag KLH presented by host MHC following in vivo immunization. ATX B10 mice grafted with FP THY/LIV also cleared Pneumocystis carinii infections, whereas simultaneously-treated ATX B10 mice not receiving FP THY were unable to do so. Discordant xenogeneic thymus grafting can therefore restore immune competence. Thus, in addition to tolerance induction, xenogeneic thymic replacement might have a potential role in the reconstitution of immunity in patients afflicted with immunodeficiencies affecting the thymus.
Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction.
Authors: Zoller K E , MacNeil I A , Brugge J S
Published: December 2022

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.
Anti-human kappa opioid receptor antibodies: characterization of site-directed neutralizing antibodies specific for a peptide kappa R(33-52) derived from the predicted amino terminal region of the human kappa receptor.
Authors: Buchner R R , Vogen S M , Fischer W , Thoman M L , Sanderson S D , Morgan E L
Published: December 2022

Site-directed polyclonal Abs specific for a synthetic peptide with sequence homology to the predicted N-terminal sequence of the human kappa opioid receptor [anti-kappa R-(33-52)] are capable of binding to normal human cells and cell lines expressing mRNA specific for the human kappa receptor. Flow cytometric analysis of 1) a neuronal cell line (NT2), 2) blood-derived CD14+ monocytes, 3) monocyte-like cell lines (U937 and THP 1), 4) blood-derived CD3+ T cells and a T cell line, and 5) human B cell lines bound anti-kappa R-(33-52) in a specific manner. Anti-kappa R-(33-52) was also found to specifically neutralize the immunosuppressive activities associated with the kappa R-selective agonist U50,488H. This antiserum was found to block U50,488H-mediated inhibition of 1) Staphylococcus aureus Cowen strain I-induced B and T lymphocyte proliferation, 2) PHA-induced T lymphocyte proliferation, and 3) S. aureus Cowen strain I-induced IgG production. However, this antiserum failed to neutralize mu R-selective agonist (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol)-mediated suppression of IgG synthesis. Finally, the kappa R-selective antagonist nor-binaltorphimine hydrochloride inhibits the binding of anti-kappa R-(33-52) to the U937 cell line. These results suggest that anti-kappa R-(33-52) specifically interacts with the human kappa R molecule. Studies conducted with anti-kappa R-(33-52) indicated that this antiserum effectively blocked U50,488H-mediated immunosuppression, but by itself did not enhance or suppress lymphocyte activation. These data suggest that anti-kappa R-(33-52) 1) does not interact with the effector binding site of the receptor, but sterically interferes with U50,488H binding to the receptor; and/or 2) the antiserum interacts with a secondary binding site that is important for ligand binding, but may not be involved in signal transduction.
Analysis of IgM structures involved in J chain incorporation.
Authors: Wiersma E J , Chen F , Bazin R , Collins C , Painter R H , Lemieux R , Shulman M J
Published: December 2022

J chain is associated with pentameric IgM and polymeric IgA. In IgM, J chain is disulfide bonded to the C575 residue of the mu-chain, located in the mu tail piece (mu tp). Previous studies indicated that mu tp is not sufficient to mediate J chain incorporation into polymeric Ig. In this study, we analyzed which other C mu domains are involved in J chain incorporation. Three altered forms of mouse IgM were analyzed: IgM lacking the C mu 1 domain, IgM in which the C mu 2 and C mu 3 domains were replaced by the hinge region and the C gamma 2 domain of IgG2b, and IgM, in which the C mu 4 domain was replaced by C gamma 3. We found that neither C mu 1, C mu 2, nor C mu 3 was absolutely required for J chain incorporation. The importance of C mu 4 could not be fully analyzed because the C gamma 3 replacement mutant failed to form polymers. Also, we found that the glycosylation site at asparagine 563 of mu tp was important for J chain incorporation. Disruption of this site by replacement of either asparagine 563 by tyrosine or serine 565 by phenylalanine resulted in diminished J chain incorporation and increased production of hexameric IgM. These results demonstrate the importance of structural elements located close to mu tp in the incorporation of J chain into IgM.