bluuebunny/crossref_metadata_2025 huggingface dataset:
The IL-2 receptor gamma c chain does not function as a subunit shared by the IL-4 and IL-13 receptors. Implication for the structure of the IL-4 receptor.
Authors: He Y W , Malek T R
Published: December 2022

The IL-2 receptor (IL-2R) gamma c subunit is also a component of the receptors for IL-4, IL-7, IL-9, and IL-15. The IL-4R and IL-13R appear to share a common subunit, and gamma c was proposed to be this shared subunit. In this study, we have assessed the relative contribution of gamma c to the mouse IL-4R and IL-13R. The MC/9 mast cell line constitutively expresses gamma c and proliferates to IL-4 and IL-13, but only the response to IL-4 was blocked by anti-gamma c mAbs. After transfection of the IL-4- and IL-13-responsive gamma c-negative B9 plasmacytoma with full length (m gamma) or cytoplasmic-tailless gamma c cDNA (m gamma t), only the proliferative response to IL-4 was affected by the surface expression of these gamma c molecules. The inability of m gamma or m gamma t expression to affect IL-13-induced proliferation by B9 indicates that gamma c does not obviously contribute to the IL-13R and does not function as the shared subunit of the IL-4R and IL-13R. This study suggests that there are two distinct IL-4R, one of which is independent of gamma c.
Antigen processing in populations of mature murine dendritic cells is caused by subsets of incompletely matured cells.
Authors: Koch F , Trockenbacher B , Kämpgen E , Grauer O , Stössel H , Livingstone A M , Schuler G , Romani N
Published: December 2022

Immature dendritic cells (DC), such as freshly isolated Langerhans cells (LC), are excellent at processing native protein Ag. During short term culture they shut off MHC class II synthesis and down-regulate their processing capacity. They retain, however, the MHC/peptide complexes, up-regulate adhesion and costimulatory molecules, and acquire the ability to sensitize T cells. Two reports describing substantial processing activity in populations of mature DC prompted us to undertake an extensive comparative study of the Ag-processing capacities of immature vs mature DC. We used a panel of 17 peptide-specific T cell hybridomas restricted by six different MHC class II molecules: I-Ab, I-A(d), I-E(d), hybrid I-A beta dE alpha, I-Ak, and I-Ek. Side by side comparisons revealed in all cases that freshly isolated LC were superior to cultured mature LC in their ability to process native proteins. With some hybridomas, however, we found a considerable degree of processing by populations of cultured LC at high doses of Ag or Ag-presenting cells. This activity, however, did not correlate with the MHC haplotype. Direct comparison over wide ranges of DC doses or Ag doses showed that it was always less than that of corresponding fresh immature LC. Immunoperoxidase staining of cytospins and flow cytometry with mAb In1 disclosed a small (20% maximum) subset of cultured LC expressing the MHC class II-associated invariant chain, indicating ongoing biosynthesis of this molecule and, thus, incomplete maturation of these LC. Therefore, the residual processing activity observed in populations of mature DC may be explained by small subpopulations of incompletely matured DC.
Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma.
Authors: Memon S A , Moreno M B , Petrak D , Zacharchuk C M
Published: December 2022

Overexpression of Bcl-2 can prevent or markedly delay cell death induced by a variety of apoptotic stimuli. Although Fas and Fas ligand (FasL) interactions play a major role in the elimination of self-reactive T cells in the periphery, inhibition of Fas-mediated killing by Bcl-2 has not been consistently observed. The mouse T hybridoma 2B4.11 (2B4) has been a useful model to study glucocorticoid- and activation-induced apoptosis, which is mediated through Fas and FasL. Using both stable transfectants and transient transfections, overexpression of Bcl-2 or Bcl-xL readily blocked glucocorticoid-induced but not activation-induced apoptosis of 2B4 cells. Bcl-2 expression did not inhibit Fas-mediated cytotoxicity triggered by cells expressing FasL or by the transient transfection of human Fas. Similarly, overexpression of Bcl-2 in the mouse T hybridoma A1.1 did not block activation-induced/Fas-mediated apoptosis. In Jurkat cells, however, expression of Bcl-2 partially inhibited anti-Fas-induced cell death. A Bcl-2-related protein that can interfere with anti-Fas killing, the adenoviral E1B 19K, also did not block activation-induced/Fas-mediated apoptosis in 2B4 cells. In contrast, expression of CrmA, a cowpox virus protein that inhibits ICE-like protease activity, blocked activation-induced apoptosis in 2B4 cells but had little effect on Dex-mediated cytotoxicity. These results show that: 1) Bcl-2 can have strikingly different anti-cell death activity in the same cell depending upon the apoptotic stimulus, 2) distinct apoptosis signaling pathways may exist with differential sensitivity to Bcl-2 and ICE-like protease inhibitors.
Induction of tolerance in freshly isolated alloreactive T cells by activated T cell stimulators is not due to the absence of CD28-B7 interaction.
Authors: Satyaraj E , Rath S , Bal V
Published: December 2022

Human T cells express MHC class II molecules on activation, and this makes them potential APCs for responder CD4 T cells. We have shown earlier that MHC class II-expressing human T cell APCs induce specific tolerance in freshly isolated alloreactive responder CD4 T cells. In this study, we show that this induction of tolerance does not depend on the expression of a specific coreceptor on the stimulator T cell APCs, because both CD4 and CD8 T cell stimulators efficiently induce tolerance. Such a tolerant cell population responds significantly better to IL-2 than unprimed cells, indicating the physical presence of T cells expressing higher levels of IL-2 receptors. The addition of exogenous IL-2 during priming with activated T cell APCs effectively blocks the induction of tolerance and permits successful priming of alloreactive responder cells. We have investigated the possible role of the CD28/CTLA4-B7 family interaction in this model of induction of T cell anergy in T-T interactions. The freshly activated T cell APCs used in this study express CTLA4-binding ligand(s). Furthermore, cross-linking CD28 on responder T cells does not enable activated T cell APCs to elicit a primary proliferative alloresponse, nor does it prevent the induction of tolerance in responder T cells. The CD28/CTLA4-B7 family interaction is thus unlikely to be involved in the induction of T cell anergy by T cell APCs.
Mapping of receptor binding sites on IL-1 beta by reconstruction of IL-1ra-like domains.
Authors: Boraschi D , Bossù P , Ruggiero P , Tagliabue A , Bertini R , Macchia G , Gasbarro C , Pellegrini L , Melillo G , Ulisse E , Visconti U , Bizzarri C , Del Grosso E , Mackay A R , Frascotti G , Frigerio F , Grifantini R , Grandi G
Published: December 2022

Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1 beta in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL-9: 127/263 R/T-->W/Y; SMIL-10: 125/127/263/265 T/R/T/Q-->R/W/Y/E; SMIL-15:222/227 I/E-->S/S) showed an increased binding capacity for IL-1RI, not paralleled by increased agonist activity, indicating that the introduced IL-1ra residues could be involved in the nonagonist IL-1RI binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1RI. Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1RI (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1RII (CDw121b) and acted as a selective antagonist of IL-1RII. From these results the following conclusions can be drawn. IL-1 beta binds to IL-1RI and to IL-1RII through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1RI. Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1RI occur through distinct sites, with loops 164-173 and 202-214 of IL-1 beta identified as two of the sites selectively involved in agonist binding to the activating receptor.
Parasite strain specificity of bovine cytotoxic T cell responses to Theileria parva is determined primarily by immunodominance.
Authors: Taracha E L , Goddeeris B M , Teale A J , Kemp S J , Morrison W I
Published: December 2022

The parasite strain specificity of CTL responses to Theileria parva varies among cattle immunized with the same parasite stock. We have investigated the influence of class I MHC on the strain specificity of CTL responses to T. parva in 19 cattle of defined class I phenotype immunized with either of two T. parva populations, in which protection to subsequent reciprocal challenge correlated with CTL strain specificity. In the majority of animals the response was restricted by the products of one MHC haplotype and there was a consistent bias to some haplotypes in preference to others. In 10 of 13 cattle expressing the molecularly defined MHC specificities A10 and KN104 on one haplotype, the CTL response was restricted entirely by this haplotype, thus allowing a precise analysis of the MHC restriction specificities. The MHC restriction specificity and the parasite population used for immunization both influenced the strain specificity of the response. By examining responses in identical twins immunized with different parasites or in animals before and after challenge with heterologous parasites, animals that mounted a strain-specific response to primary infection were shown to be capable of responding to Ags shared by the two parasite populations. These findings indicate that the strain specificity of CTL responses to T. parva is not determined primarily by immune response genes that define the inherent capacity to respond, but rather is a consequence of the response in individual animals being biased toward a limited number of immunodominant peptide-MHC determinants.
Cyclic nucleotide phosphodiesterase type IV participates in the regulation of IL-10 and in the subsequent inhibition of TNF-alpha and IL-6 release by endotoxin-stimulated macrophages.
Authors: Kambayashi T , Jacob C O , Zhou D , Mazurek N , Fong M , Strassmann G
Published: December 2022

We have recently shown that PGE2 inhibits the release of TNF-alpha from LPS-stimulated murine peritoneal macrophages via a feedback mechanism involving IL-10. Here we demonstrate that a rolipram-sensitive phosphodiesterase (PDE) type IV participates in the regulation of IL-10 synthesis. Selective PDE IV inhibitors (rolipram and RO-20-1724), but not selective inhibitors of other types of PDE, significantly augment marcrophage IL-10 production and contribute to the inhibition of TNF-alpha and IL-6 release. The addition of rolipram to LPS-stimulated macrophages results in the accumulation of cAMP and in the significant augmentation of IL-10 release. Competitive PCR analysis reveals that the drug dramatically increases IL-10 mRNA, but does not affect TNF-alpha mRNA. The inhibitory effect of rolipram on TNF-alpha can be significantly but incompletely reversed by anti-IL-10 Ab, whereas the effect of the drug on IL-6 can be completely reversed by anti-IL-10. In endotoxemic mice, the administration of rolipram increases serum IL-10 and reduces TNF-alpha and IL-6 levels. Northern blot analysis of spleens from these mice shows that rolipram increases IL-10 mRNA, whereas TNF-alpha mRNA remains largely unchanged. These results suggest that a rolipram-sensitive PDE type IV is involved in the production of IL-10 and in turn contributes to the inhibition of TNF-alpha and IL-6 release.
Identification of messenger RNA for IL-4 in human eosinophils with granule localization and release of the translated product.
Authors: Moqbel R , Ying S , Barkans J , Newman T M , Kimmitt P , Wakelin M , Taborda-Barata L , Meng Q , Corrigan C J , Durham S R , Kay A B
Published: December 2022

Human eosinophils are cytokine-producing cells that are prominent in IgE-dependent allergic tissue reactions. IL-4 promotes the development of the Th2-type phenotype in T cells and is an essential cofactor for IgE production by B cells. We detected mRNA for IL-4 by reverse transcription-PCR in blood eosinophils from atopic asthmatics. By specific ELISA, 108 +/- 20 pg of IL-4 protein/10(6) cells could be extracted from whole cells, and approximately 30% of the IL-4 was released after incubation with serum-coated particles. Using immunocytochemistry, eosinophils from atopic asthmatics and nonatopic controls showed IL-4 immunoreactivity using an anti-IL-4 mAb. IL-4 was located predominantly in the eosinophil granules, as shown by both immunogold electron microscopy and a cell fractionation technique that dissociated cell granules from membrane and cytosolic components. IL-4 mRNA colocalized with eosinophils (using sequential immunocytochemistry with an eosinophil-specific (EG2) mAb and in situ hybridization using an IL-4-specific antisense riboprobe) in both cell cytospins from bronchoalveolar lavage fluid from asthmatics as well as skin biopsies obtained from allergen-induced late phase (6-h) reactions in atopic subjects. Using double immunocytochemistry on skin biopsies with eosinophil- and IL-4-specific mAb, 83.5 +/- 3.5% of eosinophils were IL-4+. Conversely, eosinophils accounted for 46.5 +/- 3.9% of the total cells expressing IL-4 immunoreactivity. Thus, human eosinophils express mRNA for IL-4, and the translated product is contained within the crystalloid granule from which it is released after stimulation with serum-coated particles. These observations are consistent with the hypothesis that eosinophils contribute to the development of the Th2 phenotype by T cells infiltrating atopic allergic reactions as well as to IgE synthesis.
Follicular dendritic cells, interdigitating cells, and cells of the monocyte-macrophage lineage are the C1q-producing sources in the spleen. Identification of specific cell types by in situ hybridization and immunohistochemical analysis.
Authors: Schwaeble W , Schäfer M K , Petry F , Fink T , Knebel D , Weihe E , Loos M
Published: December 2022

In a mouse model, we have shown previously that macrophages are the principal source of complement C1q. Furthermore, spleen, heart, and brain were found to contain substantial levels of murine C1q-specific mRNA, whereas liver, kidney, lung, and small intestine contained only trace amounts of C1q-specific mRNA. This work addresses the identification of C1q-expressing spleen cells in the rat, using Northern blotting and in situ detection of rat C1q mRNA combined with immunohistochemical analysis. The complete sequence of mRNA encoding the B chain of rat C1q was established. The cloned cDNA was found to hybridize primarily with spleen-derived mRNA of 1.2 kb, and additionally with a novel mRNA species of 3 kb. In situ hybridization together with immunohistochemistry revealed most of the C1q-expressing cells to be located in the red pulp of the spleen, and to be mainly of the monocyte-macrophage lineage, as indicated by coexpression of ED-1, an established marker for this type of cell. In addition, C1q was expressed in S-100-positive but ED-1-negative cells, in germinal center follicular dendritic cells, and in some interdigitating dendritic cells of the periarteriolar lymphatic sheath (PALS). These results indicate that the spleen, containing the above APCs that are all involved to a major extent in the adaptive immune response and are all capable of synthesizing C1q that is involved intimately in the innate immune response, may provide the site at which the innate and adaptive immune systems merge.
B7 costimulation is necessary for the activation of the lytic function in cytotoxic T lymphocyte precursors.
Authors: Guerder S , Carding S R , Flavell R A
Published: December 2022

B7-CD28 costimulation is essential for the activation of CD4+ T helper cells, mainly by regulating IL-2 and other cytokine production. The requirement for this costimulatory pathway in the activation of CD8+ T cells, however, is still poorly understood. Here we analyzed the role of B7-CD28 costimulation in the differentiation of Ag-specific CTL precursor. We found that the activation of not only IL-2 production but also cytotoxic function in CD8 T cells requires B7 costimulation. The costimulatory signal, which cannot be replaced by exogenous IL-2, is directly implicated in the activation of the lytic machinery in CD8 T cells. Moreover, B7-CD28 costimulation appears to play a critical role in the accumulation of mRNA encoding at least one of the granzymes required for cytolytic function, granzyme B or CTLA-1. The production of IFN-gamma by CD8 T cells, however, does not appear to require costimulation.
Activation of human epidermal Langerhans cells by engagement of the high affinity receptor for IgE, Fc epsilon RI.
Authors: Jürgens M , Wollenberg A , Hanau D , de la Salle H , Bieber T
Published: December 2022

Human epidermal Langerhans cells (LCs) bind IgE via the high affinity receptor (Fc epsilon RI), and therefore are suspected to be involved in the genesis of atopic diseases. In this study, we report that surface expression of Fc epsilon RI is increased dramatically on LCs from patients with atopic dermatitis (AD) when compared with nonatopic individuals. Cross-linking of Fc epsilon RI on LCs from nonatopic individuals and patients with AD leads to a rapid tyrosine phosphorylation of several proteins, including p72, p77, and p95. However, upon receptor ligation, calcium mobilization is only detected in LCs freshly isolated from patients with AD (responder LCs), but not in those from normal skin of healthy individuals (nonresponder LCs). The beta-chain of Fc epsilon RI is not detected in normal LCs and only in a minority of LCs from atopic individuals, indicating that it is not related to the capacity of LCs to respond to Fc epsilon RI-mediated activation. In contrast, LCs from both nonatopic and atopic individuals internalize Fc epsilon RI by receptor-mediated endocytosis as a prerequisite for Ag focusing. Therefore, LCs from normal individuals and individuals with AD differ functionally by their Fc epsilon RI expression and by a distinct ability to respond to Fc epsilon RI-mediated activation.
A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor.
Authors: Müllberg J , Durie F H , Otten-Evans C , Alderson M R , Rose-John S , Cosman D , Black R A , Mohler K M
Published: December 2022

Many cytokines and soluble cytokine receptors are generated by limited proteolysis of membrane-bound precursors. We have examined the ability of the recently described metalloprotease inhibitor, TNF-alpha protease inhibitor (TAPI), and other protease inhibitors to modulate shedding. The membrane-bound forms of the ligands TNF-alpha and CSF-1, the p60 TNFR and the IL-6R, were expressed in COS-7 cells. As expected, TAPI blocked the spontaneous and PMA-induced release of TNF-alpha from transfected cells. Interestingly, TAPI also inhibited the release of soluble forms of p60 TNFR and IL-6R in COS-7 cells. However, the processing of CSF-1, which also requires proteolytic cleavage of a membrane protein, was not affected. The ability of TAPI to inhibit shedding was unique, since several other classes of protease inhibitors, including three other metalloprotease inhibitors, did not inhibit shedding of IL-6R. To determine whether TAPI would prevent shedding under more physiologic conditions, we demonstrated that TAPI was able to prevent unstimulated and PMA-induced release of the soluble forms of TNF-alpha, p60 TNFR, and IL-6R from the monocytic cell line, THP-1, and from human peripheral blood monocytes. In addition, TAPI was able to inhibit LPS-induced shedding of the p60 TNFR and TNF-alpha from monocytes. In summary, our results indicate that a metalloprotease or group of related metalloproteases is responsible for the proteolytic cleavage of several cell surface proteins.
Mouse lymphotoxin-beta receptor. Molecular genetics, ligand binding, and expression.
Authors: Force W R , Walter B N , Hession C , Tizard R , Kozak C A , Browning J L , Ware C F
Published: December 2022

Lymphotoxin (LT) -alpha beta heterotrimer is a membrane-anchored ligand expressed by activated T cells which binds specifically to the LT beta receptor (LT beta R), a member of the TNFR family. The LT beta R is implicated as a critical element in controlling lymph node development and cellular immune reactions. To address this hypothesis we have isolated a mouse cDNA encoding a single transmembrane protein of 415 amino acids with 76% identity to human LT beta R. The receptor function of this molecule was demonstrated by the ability of the extracellular domain, constructed as a chimera with the Fc region of IgG7, to bind to LT alpha beta complexes expressed on the surface of activated T cells or insect cells infected with baculoviruses containing LT alpha and LT beta cDNAs. The gene encoding mouse LT beta R, Ltbr, contains 10 exons spanning 6.9 kb and maps to mouse chromosome 6, which is closely linked to Tnfr1, consistent with the tight linkage of the human homologue of these genes on chromosome 12p13. Mouse LT beta R mRNA is expressed by cell lines of monocytic and epithelial origin but not by a CTL line, and in vivo it is constitutively expressed in visceral and lymphoid tissues. The delineation of the structure of the mouse LT beta R will aid investigations into the role of this cytokine-receptor system in immune function and development.
Molecular cloning and functional characterization of a novel member of the C-C chemokine family.
Authors: Hara T , Bacon K B , Cho L C , Yoshimura A , Morikawa Y , Copeland N G , Gilbert D J , Jenkins N A , Schall T J , Miyajima A
Published: December 2022

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. We have isolated a novel chemokine cDNA, designated CCF18, from a cDNA library of an IL-3-dependent murine pro-B cell line, Ba/F3. The cDNA encodes a protein structurally related to the C-C chemokine members. Among this family, C10 shows the highest homology to CCF18, and MIP-1 alpha also has a significant homology but to a lesser extent. CCF18 produced from COS cells induced chemotaxis and Ca2+ flux in CD4+ T cell clones. Moreover, prior administration of MIP-1 alpha desensitized the cells to CCF18. The CCF18 gene (Scya10) was mapped to a middle region of murine chromosome 11, where other genes for several C-C chemokine members are localized. These results clearly indicate that CCF18 is a new member of the C-C chemokine family. Since a high level of CCF18 mRNA is constitutively expressed in macrophage and myeloid cell lines, CCF18 may play a role in inflammatory processes.
CD66 family members are associated with tyrosine kinase activity in human neutrophils.
Authors: Skubitz K M , Campbell K D , Ahmed K , Skubitz A P
Published: December 2022

The granulocyte activation Ags, CD66a, CD66b, CD66c, and CD66d, are expressed at low levels on resting blood granulocytes, but their surface expression is up-regulated following stimulation. CD66a, in contrast to CD66b and CD66c which are anchored to the membrane via a glycosyl-phosphatidylinositol linkage, is a transmembrane protein with a cytoplasmic domain. We have previously reported that CD66a is phosphorylated in human neutrophils, largely on tyrosine, with a lower level of phosphoserine. We have now found that CD66a undergoes a rapid increase in phosphorylation following stimulation with FMLP, platelet-activating factor, and 12-O-tetradecanoyl-phorbol-13-acetate. This increase in phosphorylation was transient, with maximal phosphorylation observed by 1 min and a return to base line by 5 min following stimulation. Protein kinase activity was detected in neutrophils associated with CD66a, CD66b, and CD66c. Most of the protein kinase activity associated with these Ags was tyrosine kinase activity, with a lesser amount of threonine and serine kinase activities. Lyn and Hck accounted for much of the associated tyrosine kinase activity. The data suggest that phosphorylation of CD66a on tyrosine by an associated tyrosine kinase may play a role in the function of CD66a. In addition, associated tyrosine kinase activity may play a role in signal transduction from CD66a, CD66b, and CD66c to regulate other cell functions.
Identification of protein tyrosine phosphatase-like IA2 (islet cell antigen 512) as the insulin-dependent diabetes-related 37/40K autoantigen and a target of islet-cell antibodies.
Authors: Bonifacio E , Lampasona V , Genovese S , Ferrari M , Bosi E
Published: December 2022

The majority of patients with insulin-dependent diabetes (IDDM) have Abs to 40- and/or 37-kDa tryptic fragments (37/40K-Abs) deriving from an unidentified islet cell membrane protein distinct from glutamate decarboxylase (GAD). Recently, autoantibodies against ICA512, which has identity with the protein tyrosine phosphatase-like protein IA2, were reported. In this study we have examined whether IA2/ICA512 is the Ag specificity of 37/40K-Abs, and one of the determinants of islet cell Abs (ICA) detected by immunofluorescence. Serum from 51 of 100 new onset IDDM patients immunoprecipitated 40- and/or 37-kDa insulinoma polypeptides, and 53 immunoprecipitated in vitro translated rIA2; 49 had both 37/40K-Abs and rIA2 Abs. There were strong correlations between the levels of Abs to rIA2 and both 40 kDa (r = 0.85, p < 0.0001) and 37 kDa (r = 0.70, p < 0.0001) insulinoma polypeptides. Trypsin treatment of immunoprecipitated rIA2 yielded 40- and 37-kDa fragments, and preincubation of sera with rIA2 completely inhibited binding to the insulinoma 40- and 37-kDa polypeptides. IA2 Ab levels also correlated with ICA titer in GAD-Ab negative sera, and preincubation with rIA2 reduced ICA staining intensity in sera with ICA and IA2 Abs, but not in sera with ICA in the absence of IA2 Abs. These results provide clear evidence for the identification of IA2/ICA512 as the precursor of the islet 40- and 37-kDa polypeptide autoantigens and as one of the ICA specificities. Combined detection of Abs to IA2 and GAD65 in a single radio-binding assay identified Abs in 88 of 100 IDDM patients, and potentially facilitates population screening for IDDM risk assessment.
Frequency of molecular mimicry among T cell peptides as the basis for autoimmune disease and autoantibody induction.
Authors: Garza K M , Tung K S
Published: December 2022

Experimental ovarian autoimmune disease is inducible by immunization with an ovarian peptide from zona pellucida (ZP) 3, a glycoprotein of the zona pellucida on mouse oocyte. The murine ZP3 peptide contains two nested T cell epitopes with slightly different critical residue motifs. To investigate the frequency of cross-reaction between nonovarian and ovarian peptides, we have chosen arbitrarily 16 nonovarian peptides with complete or partial homology to the critical residue motifs of the two T cell epitopes. Based on peptide induction of autoimmune oophoritis and T cell-dependent amplified autoantibody response to ovarian ZP, cross-reaction was documented for 7 (or 44%) of the 16 nonovarian peptides studied. Thus, molecular mimicry as a pathogenetic mechanism of autoimmunity should not be limited by the frequency of cross-reaction among self and nonself peptides. On the other hand, the sharing of critical residue motif per se is not sufficient for mimicry to occur, nor does it predict peptide cross-reaction.
Molecular mapping of a pathogenically relevant BP180 epitope associated with experimentally induced murine bullous pemphigoid.
Authors: Liu Z , Diaz L A , Swartz S J , Troy J L , Fairley J A , Giudice G J
Published: December 2022

Bullous pemphigoid (BP) and herpes gestationis (HG) are subepidermal blistering diseases associated with an autoimmune response directed against BP180, an epidermal hemidesmosomal glycoprotein. The pathogenic relevance of this Ag/Ab system was established by the recent demonstration that IgG Abs reactive with the murine form of BP180 (mBP180) are capable of triggering a subepidermal blistering disease after passive transfer into neonatal BALB/c mice. The aim of the present study was to determine the fine specificity of the pathogenically relevant Abs in this experimental model of BP. Four high titer rabbit-anti-mBP180 antisera were included in this analysis--only two of which exhibited pathogenic activity in the passive transfer model. Immunoblot analysis using a panel of mBP180 deletion mutants revealed that each of the four rabbit sera reacted with at least three distinct sites on the mBP180 ectodomain; however, this technique failed to distinguish between the reactivity patterns of the pathogenic and nonpathogenic sera. An alternative technique, liquid phase immunoadsorption analysis, was used to identify one mBP180 antigenic site, comprising 9 to 12 amino acids and designated mBP1, that was specifically recognized by the two pathogenic sera. Pre-adsorption of pathogenically active IgG preparations with fusion proteins containing the mBP1 antigenic site resulted in complete blocking of immunofluorescence reactivity with the murine basement membrane zone (BMZ) and in complete neutralization of pathogenic activity. Anti-BMZ reactivity displayed by nonpathogenic Abs was not altered or diminished by pre-adsorption with this same mBP180 recombinant protein. These findings should help to elucidate the immunopathologic mechanisms responsible for human BP and HG and may have significant implications in the diagnosis and treatment of these autoimmune diseases.
Activated adhesion of CTL to MHC class I but not to fibronectin is inhibited by cis unsaturated fatty acids and phenylarsine oxide.
Authors: Anel A , Mescher M F , Kleinfeld A M
Published: December 2022

Binding of CTL to MHC class I or fibronectin is activated through TCR signaling. Once activated, CTL adhesion to MHC class I results in tyrosine phosphorylation of CTL substrates, phosphatidylinositol (PI) turnover, and degranulation. Although activated adhesion to fibronectin does not itself initiate PI hydrolysis or degranulation, these responses are amplified once they become activated. In the present study we have examined the effect of cis unsaturated fatty acids (FA) and phenylarsine oxide (PAO) on CD8-mediated adhesion of CTL to immobilized class I protein and on biochemical and functional events that are triggered by this adhesion. Previous studies have shown that FA and low concentrations of PAO inhibit specific tyrosine phosphorylation events and degranulation but have no effect on PI turnover or CTL-target cell conjugates. The present results show that pretreating CTL with cis unsaturated, but not saturated, FA and low concentrations of PAO (< 0.5 microM) inhibit soluble anti-TCR-triggered binding of CD8 to immobilized MHC class I, tyrosine phosphorylation of CTL substrates, PI turnover, and degranulation. Addition of cis unsaturated FA or PAO after CTL have been allowed to bind to immobilized class I protein did not affect the level of adhesion. In contrast, neither cis unsaturated FA nor PAO affected the TCR-activated binding of CTL to fibronectin. These results suggest that activation of adhesion to the class I and fibronectin ligands involves divergent different pathways that can be distinguished by the FA and PAO agents.
Selection for S107-V11 gene expression by peritoneal B cells in adult mice.
Authors: Pennell C A
Published: December 2022

Peritoneal B-1 cells in adult mice express a restricted repertoire of V genes. To determine the basis for the nonrandom expression of one VH gene (the V11 gene in the S107 family), and to determine if this gene is nonrandomly expressed early in development, we amplified rearranged S107 genes in adult and neonatal tissues taken from B10.H-2aH-4bp/Wts (2a4b) mice. In adult peritoneal B cells, the S107-V11 gene was found to be productively rearranged up to five times more frequently than V1, another member of the S107 VH gene family. This did not occur in adult spleen, or in neonatal spleen or liver. In these three tissues the productive S107-V11/V1 rearrangement ratios ranged from 0.5 to 0.9. This difference suggests that S107-V11-expressing peritoneal B cells in adult mice have been selected and clonally expanded. Clonal expansion is further suggested by the differing ratios of productive to nonproductive rearrangements. In adult peritoneal B cells, the S107-V11 productive/non-productive ratio is higher than that of V1, consistent with the clonal expansion of S107-V11-expressing cells but not of V1-expressing cells. Analysis of the VHDJH junctional sequences in the productive S107-V11 rearrangements reveals that there are no predominant restrictions in the lengths or sequences of the third complementarity determining regions (CDR3). Selection is therefore independent of CDR3 and so must be based on germline-encoded portions of the S107-V11 region.
Identification of recombinant filarial proteins capable of inducing polyclonal and antigen-specific IgE and IgG4 antibodies.
Authors: Garraud O , Nkenfou C , Bradley J E , Perler F B , Nutman T B
Published: December 2022

Filarial infection is characterized by an immune response associated with the production of Ag-specific IgG4 and IgE and IL-4 and IL-5. To identify filarial Ags capable of inducing such responses and to analyze the role Ags themselves play in sustaining it, 24 recombinant filarial parasite proteins were screened for their ability to be recognized by sera from 67 individuals with tissue-invasive filarial infections. Among the recombinant proteins that were recognized by IgG4 or IgE Abs in 25% of the sera or more, two were selected on the basis of their ability to elicit polyclonal and Ag-specific IgE/IgG4 Abs in vitro. Ov27 (analogous to Ov7/cystatin, a cysteine protease inhibitor) and OvD5B (analogous to Ov33, an aspartyl protease inhibitor) induced both a polyclonal and Ag-specific IgE/IgG4 response that was blocked by neutralizing Abs to IL-4 and to IL-13 or by soluble IL-4 receptors. Recombinant human IFN-gamma and IL-12 also led to a decrease in the production of polyclonal and Ag-specific IgE/IgG4 Abs. In addition, these two recombinant proteins preferentially stimulated the secretion of IL-4, IL-5, and IL-10 (in contrast to IFN-gamma). The data suggest that certain epitopes on filarial Ags preferentially elicit a Th2-type response and provide an in vitro model for dissecting the mechanisms underlying this preferential response.
Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva.
Authors: Toye P G , Metzelaar M J , Wijngaard P L , Nene V , Iams K , Roose J , Nyanjui J K , Gobright E , Musoke A J , Clevers H C
Published: December 2022

Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5' and 3' regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3' region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.
The serine/threonine phosphatase inhibitor, calyculin A, inhibits and dissociates macrophage responses to lipopolysaccharide.
Authors: Barber S A , Perera P Y , McNally R , Vogel S N
Published: December 2022

LPS-stimulated macrophages (M phi) produce inflammatory mediators that are largely responsible for the pathophysiology associated with septic shock. M phi respond to LPS with rapid protein phosphorylation and dephosphorylation on serine, threonine, and tyrosine residues. If these events are critical for the cellular response to LPS, the kinases and/or phosphatases involved may be vulnerable targets for pharmacologic intervention. Recent studies demonstrated that tyrosine kinase inhibitors block LPS-induced tyrosine phosphorylation of MAP kinases as well as TNF-alpha and IL-1 beta production. To investigate a role for serine/threonine phosphatases, we evaluated the effect of calyculin A, a potent serine/threonine phosphatase inhibitor, on LPS stimulation of murine M phi. Pretreatment of M phi with calyculin A inhibited LPS-induced expression of six immediate-early genes: TNF-alpha, IL-1 beta, IFN-beta, IP-10, IRF-1, and TNFR-2. Calyculin A added 1.5 h after LPS treatment greatly reduced accumulation of IP-10, IRF-1, and TNFR-2 mRNA, but not TNF-alpha, IL-1 beta, and IFN-beta mRNA. Calyculin A, in the absence or presence of LPS, resulted in sustained tyrosine phosphorylation of the MAP kinases. These findings suggest that an "early" serine/threonine phosphatase activity is essential for LPS stimulation of M phi and that the activation of MAP kinases is not sufficient for the induction of these immediate-early genes. The requirement for a "late" phosphatase activity for expression of a subset of LPS-inducible genes dissociates at least two regulatory pathways in LPS signal transduction.
Enhancement by the complement membrane attack complex of tumor necrosis factor-alpha-induced endothelial cell expression of E-selectin and ICAM-1.
Authors: Kilgore K S , Shen J P , Miller B F , Ward P A , Warren J S
Published: December 2022

Although TNF-alpha and several products of the activated complement system (e.g., C3b, iC3b, and C5a) are known to modulate endothelial cell function in vitro, relatively little is known about the potential modulatory role of the membrane attack complex (MAC) in endothelial cell activation. Using an in vitro neutrophil-endothelial adhesion assay and a quantitative whole cell ELISA to measure endothelial E-selectin and intracellular adhesion molecule-1 (ICAM-1) expression, we examined the modulatory role of the MAC in TNF-alpha-induced neutrophil-endothelial cell adhesive interactions. Activation of quiescent human umbilical vein endothelial cells (HUVECs) with TNF-alpha results in a concentration-dependent increase in neutrophil adhesion measured at 4 h. Assembly of sublytic concentrations of the MAC on endothelial cells did not result in changes in neutrophil-HUVEC adhesion measured at 4 h. Activation of HUVECs with TNF-alpha followed by assembly of the MAC resulted in a marked increase in neutrophil binding as compared with that observed in cells treated with TNF-alpha alone. Blocking studies of mAb revealed that in either TNF-alpha-stimulated or TNF-alpha and MAC-activated endothelial cells enhanced neutrophil binding was nearly entirely attributable to E-selectin and ICAM-1. This conclusion was further supported by a whole-cell ELISA, which provided evidence that the MAC augments TNF-alpha-induced up-regulation of both E-selectin and ICAM-1. This study provides data that support the conclusion that the distal complement system (MAC) can enhance TNF-alpha-induced proinflammatory endothelial cell functions.
Expression, purification, and characterization of the recombinant proform of eosinophil granule major basic protein.
Authors: Popken-Harris P , McGrogan M , Loegering D A , Checkel J L , Kubo H , Thomas L L , Moy J N , Sottrup-Jensen L , Snable J L , Kikuchi M T
Published: December 2022

The cDNA for the highly toxic eosinophil granule major basic protein (MBP) encodes a 25-kDa acidic precursor (proMBP) that is processed to form the 14-kDa mature MBP. To characterize the biochemical and biological properties of proMBP, and compare these to the known properties of MBP, we expressed recombinant proMBP in Chinese hamster ovary cells and purified the secreted form from supernatants. We developed a mAb specific for proMBP, J163-15E10, and by using a proMBP-specific RIA we found that recombinant proMBP was expressed quite efficiently at levels between 10 and 100 mg/l. By SDS-PAGE and immunoblotting analyses of bulk Chinese hamster ovary supernatants, recombinant proMBP was electrophoretically heterogeneous with an apparent molecular mass ranging from 3 x 10(4) to 1 x 10(5) daltons. Despite difficulties encountered because of the extreme molecular heterogeneity of the proform, two methods for purification of a predominant 33-kDa form of recombinant proMBP are presented. Glycosylation analysis of purified 33-kDa proMBP indicated that approximately 5 kDa is likely accounted for by the addition of one glycosaminoglycan group, three O-linked, and one N-linked complex type carbohydrate groups. Functional studies of purified recombinant proMBP were also conducted. Using amounts of proMBP determined to be optimal for MBP activity, it was shown that proMBP not only lacked the ability to inhibit protein synthesis in K562 cells, but it also lacked the ability to stimulate basophil histamine release or generate neutrophil superoxide anion release. Furthermore, proMBP inhibited in a dose-responsive manner the basophil histamine release and superoxide anion generation stimulated by MBP. The development of a mAb and RIA specific for proMBP will now make it possible to analyze biologic fluids for the presence of this protein, especially in pregnancy, when proMBP is increased.
Analysis of V beta 8.2 CDR3 sequences from spinal cord T cells of Lewis rats vaccinated or treated with TCR V beta 8.2-39-59 peptide.
Authors: Buenafe A C , Vainiene M , Celnik B , Vandenbark A A , Offner H
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE) in the Lewis rat can be induced with the administration of Gp-BP. This disease appears to be mediated at least in part by V beta 8.2+CD4+T cells, which specifically recognize the BP72-89 encephalitogenic peptide. Treatment or protection with V beta 8.2 CDR2 39-59 peptide can suppress or prevent clinical signs of EAE, presumably through the activation of regulatory T cells. Interestingly, V beta 8.2+ T cells continue to persist in the spinal cord of protected animals, although their appearance in the central nervous system (CNS) is delayed when compared with control animals with EAE. As part of our effort to elucidate the mechanism(s) of peptide protection and therapy, we sought to determine whether the V beta 8.2+ T cells in the spinal cord of protected or treated rats were truly representative of those found in rats with clinical EAE. Therefore, we examined the following CNS samples for the Asp96Ser97 motif, which has been identified previously in V beta 8.2+ BP-specific, encephalitogenic T cell clones: 1) rats protected with V beta 8.2-39-59 peptide, 2) rats treated with V beta 8.2-39-59 peptide, and 3) control rats with EAE. Our findings indicate that EAE-associated V beta 8.2+ sequences can still be found in both peptide-treated and peptide-protected rats. It appears that administration of V beta 8.2 CDR2 peptide does not prevent EAE-associated V beta 8.2+ T cells from infiltrating the CNS and that other mechanisms are at work to prevent the development of EAE.
Acute graft-versus-host reaction can be aborted by blockade of costimulatory molecules.
Authors: Hakim F T , Cepeda R , Gray G S , June C H , Abe R
Published: December 2022

Injection of parental lymphocytes into an unirradiated adult F1 host results in an acute GVH reaction characterized by immune deficiency, attack on host lymphohematopoietic tissues, and repopulation with donor-derived cells. All of these events result from the initial activation of donor lymphocytes by host alloantigens. Interaction of pairs of host and donor costimulatory molecules, in particular CD28/CTLA4 and B7-1/B7-2, play a crucial role in this initial activation of donor T cells. We demonstrate here that in vivo treatment of the host with high doses of CTLA4-Ig solely during the initial period of donor alloactivation can completely abort the subsequent development of GVH reaction. Although donor T cells are retained, CTLA4-Ig treatment reduces the initial endogenous cytokine production and arrests the subsequent expansion of donor T cells, the differentiation of anti-host effectors, and the development of severe immune deficiency. This result is consistent with the establishment of host-specific tolerance in the donor population, while maintaining host immune competence.
Ly-6C is a marker of memory CD8+ T cells.
Authors: Walunas T L , Bruce D S , Dustin L , Loh D Y , Bluestone J A
Published: December 2022

This study examined long-term phenotypic and functional effects of TCR ligation in vivo. Flow cytometric analysis of T cells from mice treated with anti-CD3 revealed an increase in CD44 expression in both the CD4+ and CD8+ populations. The phenotypic changes were a result of TCR engagement, because treatment with staphylococcal enterotoxin B (SEB) resulted in a preferential increase in CD44 expression on the SEB-reactive V beta 8 T cells. In addition, the percentage of cells expressing Ly-6C increased among the CD8+ subset after anti-CD3 treatment and in the V beta 8+ CD8+ subset after treatment with SEB. Finally, the TCR transgenic (Tg) mouse strain 2C was used to confirm that the phenotypic changes can be induced by exposure to a physiologic ligand (H-2Ld). Before treatment, nearly all of the Tg+CD8+ cells were CD44low/Ly-6C-. Tg+ peritoneal exudate T cells isolated from mice challenged with P815 cells (H-2Ld) up-regulated Ly-6C and secreted higher levels of IFN-gamma on a per Tg+ CD8+ T cell basis after treatment. Taken together, these data indicate that in vivo TCR/CD3 engagement results in phenotypic and functional changes in T cells. Furthermore, Ly-6C expression correlates with an increase in IFN-gamma production after antigenic stimulation of CD8+ T cells, suggesting that it is a "memory" marker that correlates with Ag-specific functional changes in CD8+ T cells.