bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure-function studies on a polyreactive (natural) autoantibody. Polyreactivity is dependent on somatically generated sequences in the third complementarity-determining region of the antibody heavy chain.
Authors: Martin T , Crouzier R , Weber J C , Kipps T J , Pasquali J L
Published: December 2022

SMI is a previously characterized IgM kappa polyreactive (natural) autoantibody. The variable regions of the heavy and light chains of SMI are respectively encoded by a nonmutated VH1 gene, designated 51p1, and a conserved nonmutated V kappa gene, designated Humkv325. These V genes seem to be over-represented in the autoimmune and fetal B cell repertoires, and to be frequently expressed in malignant B cells during certain lymphoid proliferations such as chronic lymphocytic leukemia. Polyreactive natural autoantibodies are thought to rely mainly on the use of such V genes in germ-line configuration. However, this model underestimates the contribution of the somatically generated heavy chain third complementarity-determining region (HCDR3) to autoantibody specificity. We used oligonucleotide site-directed mutagenesis to permute the sequence of the SMI-HCDR3 to generate a family of mutant proteins, each of which differed from the original SMI-IgM kappa by one amino acid residue. This allowed us to examine the relative contribution of selected amino acid residues in this region to the binding affinity of SMI against a panel of self-Ags. We found that a single amino acid substitution within the HCDR3 could dramatically alter the specificity of this autoantibody. Some substitutions abrogated the reactivity with all the tested Ags, whereas others changed the affinity or spectrum of reactivity for certain self-Ags. These results demonstrate that the autoantibody-binding activity of these conserved autoantibody-associated germ-line V genes is dependent upon heavy chain junctional sequences that are generated somatically during Ig gene rearrangement.
The fifth domain of beta 2-glycoprotein I contains a phospholipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies.
Authors: Hunt J , Krilis S
Published: December 2022

We have identified a phospholipid binding site in the fifth domain of beta 2-glycoprotein I (beta 2-GPI). Using synthetic peptides spanning the fifth domain of beta 2-GPI, we have shown that the presence of the sequence Glu274-Cys288 caused a decrease in the binding of purified anticardiolipin (aCL) antibodies in a modified cardiolipin (CL)-ELISA by inhibiting the binding of beta 2-GPI to CL. This peptide bound to and could be eluted from a CL affinity column in a manner similar to native beta 2-GPI. Peptides corresponding to other regions of the fifth domain had no inhibitory effect. The inhibitory activity was restricted to the sequence Cys281-Lys-Asn-Lys-Glu-Lys-Lys-Cys288. Peptides in which the two flanking cysteine residues were deleted or substituted with serine residues possessed no inhibitory activity, indicating that the conformation of this highly positively charged sequence may be critical for phospholipid binding. aCL antibodies purified from patients with autoimmune disease were shown to bind directly to wells coated with native beta 2-GPI but not to wells coated with a preparation of beta 2-GPI cleaved between Lys317 and Thr318. The integrity of this sequence is therefore critical for these antibodies to recognize beta 2-GPI, and the putative epitope for aCL antibodies is most likely to be in this region.
Direct sequence identification and kinetic analysis of an MHC class I-restricted Listeria monocytogenes CTL epitope.
Authors: Pamer E G
Published: December 2022

Murine infection with the intracellular bacterium Listeria monocytogenes elicits MHC class I-restricted CTL with specificity for multiple bacterial peptides. The variety and relative abundance of self-peptides bound by MHC molecules make identification of pathogen-derived peptides difficult. In this report, the sequence of a pathogen-derived CTL epitope is determined by direct analysis of peptides extracted from MHC class I molecules. The epitope, p60 217-225, is presented to L. monocytogenes-specific CTL by the H-2Kd MHC class I molecule and is derived from p60, a secreted invasion-associated protein. Quantitation of p60 217-225 in infected cells shows that this epitope is detectable within 2 h of infection and, after a 9-h infection, there are over 3000 epitopes per infected cell. This contrasts with listeriolysin 91-99, the other major L. monocytogenes epitope, which is present in quantities below 200 epitopes per cell until 5 h of infection and reaches 800 epitopes per cell 9 h after infection. This report shows that identifying new T lymphocyte epitopes by direct sequence analysis of peptides isolated from MHC molecules is feasible. Furthermore, kinetic and quantitative analysis of T cell epitopes in infected cells is a useful approach to investigate the multispecific CTL response to complex intracellular pathogens.
Production of mouse V/human C chimeric kappa genes by homologous recombination in hybridoma cells. Analysis of vector design and recombinant gene expression.
Authors: Sun W , Xiong J , Shulman M J
Published: December 2022

Homologous recombination between transferred and chromosomal Ig genes in mouse hybridoma cells offers a general method of altering the chromosomal Ig genes in predetermined ways. Recombination is infrequent in hybridoma cells, and we have been interested in improving the methods for identifying and recovering the rare recombinants. We have used vectors that are designed to replace the mouse chromosomal C kappa segment with the human equivalent, so that recombinants produce mouse V/human C chimeric kappa-chains. We describe an enhancerless, replacement type vector that can be used with the herpes thymidine kinase counterselection to provide such enrichment that homologous recombinants constitute 15% of the selected G418-resistant, FIAU-resistant cells. We have also measured the level of chimeric kappa gene expression and found surprisingly that (1) it is very variable among transformants with the same recombinant gene structure, (2) there is no systematic difference in the level of production by recombinants that retain or have lost the J-C kappa intron enhancer, and (3) the amount of chimeric kappa mRNA in even the highest producing transformants is much less than the amount of the corresponding mouse kappa mRNA.
Substance P enhances the secretion of tumor necrosis factor-alpha from neuroglial cells stimulated with lipopolysaccharide.
Authors: Luber-Narod J , Kage R , Leeman S E
Published: December 2022

The neuropeptide, substance P (SP), can stimulate secretion of TNF-alpha from macrophages. Neuroglia have SP receptors and subserve various macrophage-like functions in the central nervous system. We investigated whether SP stimulates secretion of TNF-alpha from primary cultures of neuroglial cells containing both astrocytes (approximately 90%) and microglia (approximately 10%). SP alone had no effect; however in the presence of LPS (10 ng/ml), SP (1 to 10 nM) caused a dose-dependent increase in TNF-alpha secretion above the level measured in response to LPS alone. The effective doses of SP correlated with 125I-labeled Bolton Hunter-conjugated SP binding (Kd 0.2 nM) to these cultures. Incubation with LPS did not change the number or affinity of SP-binding sites. In cultures enriched for microglia (> 99% pure), LPS stimulated the secretion of TNF-alpha but SP caused no enhancement. Microglia have no detectable 125I-labeled Bolton-Hunter-conjugated SP binding sites in the presence or absence of LPS. These results indicate that the action of SP is mediated through astrocytes. We investigated whether IL-1 mediates the SP enhancement of TNF-alpha secretion. Addition of IL-1-neutralizing antisera to mixed cultures stimulated with both LPS and 10 nM SP decreased TNF-alpha secretion to the level observed with LPS alone. LPS alone stimulated the secretion of IL-1 in a dose-dependent manner in the primary cultures, and this LPS-mediated IL-1 secretion was enhanced by SP. This enhancement was not observed in microglial cultures. SP may therefore play a role in neuropathologies in which these cytokines have been implicated.
Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus-induced demyelinating disease. V. Mapping of a dominant immunopathologic VP2 T cell epitope in susceptible SJL/J mice.
Authors: Gerety S J , Karpus W J , Cubbon A R , Goswami R G , Rundell M K , Peterson J D , Miller S D
Published: December 2022

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a relevant mouse model of multiple sclerosis. Demyelination is linked to persistent TMEV infection of the central nervous system and characterized by perivascular inflammatory mononuclear infiltrates and primary demyelination. Myelin damage is a T cell-dependent process and susceptibility correlates with the temporal development of chronic virus-specific delayed-type hypersensitivity (DTH) responses. Our previous results have shown that inflammatory processes mediated by Th1 cells specific for a determinant(s) on virus capsid protein 2 (VP2) play a major immunopathologic role in SJL/J mice. This study identifies a 13 amino acid peptide on VP2 (VP2(74-86)) as the immunodominant T cell epitope in TMEV-infected and -immunized SJL/J mice, and demonstrates the ability of that sequence to prime for the majority of the SJL/J DTH T cell response to intact TMEV. The importance of T cell responses to this epitope in the demyelinating process was illustrated by experiments in which SJL/J mice displayed an increased incidence and accelerated onset of clinical disease after peripheral immunization with a fusion protein containing VP2(74-84) before intracerebral infection with a suboptimal dose of the BeAn strain of TMEV. Identification of this immunopathologic TMEV T cell epitope will be critically important for delineation of the mechanisms of T cell-mediated myelin damage and for potential use to prevent and/or treat TMEV-induced demyelinating disease via the induction of epitope-specific tolerance.
Induction of IL-6 gene expression in Kaposi's sarcoma cells.
Authors: Yang J , Hagan M K , Offermann M K
Published: December 2022

IL-6 is a multifunctional cytokine that functions as an autocrine growth factor for AIDS-derived Kaposi's sarcoma (KS) cells. We report that IL-6 is highly inducible at both the mRNA and protein levels in cultured KS cells by multiple agents, yet the effect of the IL-6 on the proliferation of KS cells is dependent on the agent responsible for its induction. Both IL-1 beta and the synthetic dsRNA, poly (I:C), induced high levels of IL-6 mRNA and protein expression, whereas LPS and TNF-alpha led to only modest increases in IL-6 protein and mRNA. When KS cells were incubated with poly (I:C) in combination with either IL-1 beta or TNF-alpha, there was a synergistic increase in the level of IL-6 production, whereas LPS and TNF-alpha in combination led to only an additive increase in the level of IL-6 production. Exogenous IL-6 was shown to induce proliferation in KS cells, yet there was a dramatic inhibition of proliferation in response to poly (I:C), despite the high levels of IL-6 produced. This inhibition of proliferation by poly (I:C) was unlikely as a result of expression of class I IFN in response to the poly (I:C) because high concentrations of exogenous IFN-alpha had no demonstrable effect on [3H]TdR incorporation under conditions in which poly (I:C) caused a 90% decrease in [3H]TdR incorporation. Pretreatment of KS cells with poly (I:C) for 24 h followed by removal of the poly (I:C) led to high levels of IL-6 secreted into medium that induced proliferation in KS cells. These data suggest that in vivo, multiple agents that occur in response to infection and systemic disease could induce IL-6 production by KS cells, yet the ability of the IL-6 to influence proliferation of KS cells is dependent on the context in which the IL-6 is induced.
Ligation of CD45 on B cells can facilitate production of secondary Ig isotypes.
Authors: George A , Rath S , Shroff K E , Wang M , Durdik J M
Published: December 2022

The possibility that isotype switching in B cells may be affected by engagement of the CD45 molecule on B cells has been investigated in microcultures containing limiting numbers of B cells and nonlimiting numbers of both alloreactive Th cells and purified dendritic cells (DC). Addition of Abs to the B cell-specific isoform, B220, to the microcultures leads to an increase in the proportion of B cell clones that secrete secondary Ig isotypes. In the presence of anti-CD45 Ab, microculture wells show a 39% frequency of secondary isotypes (560/1440) compared with a 11% frequency in control microcultures (89/780). Cross-linking appears to enhance this effect. Even in cultures of B cells and DC without T cells, addition of anti-B220 induces isotype switching in a significant number of microwells. Cross-linking and capping B220 molecules results in co-capping of surface Ig and MHC class II molecules. The results suggest that signal transduction through the CD45 molecule may affect pathways involved in isotype switching.
Fas antigen is the major target molecule for CD4+ T cell-mediated cytotoxicity.
Authors: Stalder T , Hahn S , Erb P
Published: December 2022

Activation of the Fas cell surface molecule, either by specific antibody or by its as yet unidentified ligand, has been shown to induce apoptosis. Because apoptosis is also evoked in target cells by cytolytic T cells, we investigated whether the Fas pathway is involved in CD4+ T cell-mediated cytotoxicity. Analysis of Fas expression in APC, such as the B lymphoma A20.2J and MHC class II-transfected fibroblasts RT2.3, revealed a correlation between the degree of expression and sensitivity to cytotoxic attack, high level of Fas expression in A20.2J being associated with efficient lysis. To examine whether increased Fas expression in RT2.3 would render these cells more susceptible to CD4+ CTL lysis, they were transfected with a Fas gene expression vector. Indeed, Fas- but not mock-transfected RT2.3 proved to be more sensitive to lysis by either Ag specifically or nonspecifically activated CD4+ CTL. Similarly, MHC class II-negative, Fas-transfected L1210 leukemia cells were lysed with nonspecifically activated CD4+ CTL. The importance of the Fas engagement in CD4+ CTL-mediated cytotoxicity is further substantiated by the failure of both cloned and normal CD4+ CTL to lyse B cell blasts from Ipr mice. These mice are known to have a defect in functional Fas expression. Although the bulk of CD4+ T cell-mediated lysis appears to be Fas induced, the fact that the effector phase of A20.2J lysis is only partially Ca2+ independent indicates that other pathways also contribute to target cell death.
Mapping of amino acid residues in the C mu 3 domain of mouse IgM important in macromolecular assembly and complement-dependent cytolysis.
Authors: Arya S , Chen F , Spycher S , Isenman D E , Shulman M J , Painter R H
Published: December 2022

By analyzing the effects of single site mutations of a TNP-binding mouse IgM we have identified amino acid residues clustered in two regions in the C mu 3 domain that are important in the complement-dependent cytolytic activity of polymeric IgM. Some of the mutations also impaired IgM polymerization. For one of these clusters, D432G, P434A, and P436S, which lies on the fy2 and fy3 strands and their connecting loop, polymerization was little affected and the effect on the cytolytic activity of the polymer fraction was taken to imply direct involvement of the residue in C1 binding. The other cluster, involving residues D356A K361A and D417G, is situated at the other end of the C mu 3 domain closer to the center of the Fc mu disc. The D356A K361A and D417G mutations significantly impaired polymer formation, suggesting that these residues are necessary for proper folding or packing of the C mu 3 domains and may affect cytolysis only indirectly. Some other mutations had little or no effect on polymerization or cytolytic activity (E423A, E527G), whereas some mutations impaired only IgM polymerization without affecting cytolytic activity (D344A, K361A, K443A P544G). In others the defect in polymerization was so profound that only the monomer formed (H430A/N/Q and K438G). Our results also suggest that the C1 binding site of IgM is not strictly homologous to the C1 binding site of IgG. Although mutation of E318 of IgG has been shown to reduce its cytolytic activity, mutation of the homologous residue in IgM, E423, was without effect as were mutations of other flanking-charged residues. Proline at 436 in IgM and 331 in IgG may, however, be a common element.
Effects of IL-12 on the response and susceptibility to experimental viral infections.
Authors: Orange J S , Wolf S F , Biron C A
Published: December 2022

Effects of administered IL-12 on immune responses to viral infection were evaluated. IL-12-mediated effects during lymphocytic choriomeningitis virus (LCMV) infection in C57BL/6 mice were contrasted to those in either uninfected mice, or mice infected with a virus inducing a more modest T cell response, murine cytomegalovirus. Mice received 0, 1, 10, 100, or 1000 ng/d of IL-12 on the day prior to infection and daily on day 0 to 6 post-infection. Responses were examined on day 7. Low doses of IL-12 enhanced immunity to LCMV infection as demonstrated by increased splenic CD8+ T cell numbers and decreased LCMV replication. Low doses of IL-12 also significantly enhanced splenic CD8+ T cell numbers in murine cytomegalovirus-infected mice. In contrast, high doses of IL-12 were detrimental to resistance against LCMV infection. Higher doses of IL-12: 1) inhibited virus-specific CTL lytic capacity per spleen by > 90%; 2) inhibited virus-induced expansion of CD8+ T cells in spleen, peripheral blood, and lymph nodes by > 80%; 3) induced necrotic lesions in splenic white pulp; and 4) resulted in 2 log increases in splenic and renal viral replication. Initiation of high dose IL-12 treatment on day 0, 1, 2, or 4 post-infection had similar effects. Although serum levels of IFN-gamma and TNF were normally not detectable in LCMV-infected mice, infection acted synergistically with IL-12 for cytokine induction. The IL-12-mediated reduction in CD8+ T cell numbers and synergistic enhancement of circulating cytokines levels observed during LCMV infection were unique to this infection. These results demonstrate that IL-12-mediated effects can be either protective or detrimental, depending upon the environment in which the factor is presented.
Macrophage inflammatory protein-1 alpha and monocyte chemoattractant peptide-1 elicit immediate and late cutaneous reactions and activate murine mast cells in vivo.
Authors: Alam R , Kumar D , Anderson-Walters D , Forsythe P A
Published: December 2022

We have previously reported that monocyte chemoattractant protein-1 (MCP-1) is the most potent histamine-releasing factor (HRF) for basophils. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has modest histamine-releasing activity. The objective of this study was to investigate whether MCP-1 and MIP-1 alpha would activate mast cells in vivo and induce a cutaneous inflammatory reaction in mice. To this goal, mouse hind footpads were separately injected with 20 microliters of human recombinant MCP-1 or MIP-1 alpha (10(-7) M). Diluent was used as a control in the second footpad. The footpad-swelling response was measured at 30 min, 1 h, and then hourly for 6 h. Both MCP-1 (2.72 +/- 0.2 vs 2.1 +/- 0.03 mm for diluent, n = 8, p < 0.02) and MIP-1 alpha (3.0 +/- 0.1 vs 2.1 +/- 0.03 mm for diluent, n = 8, p < 0.02) induced an immediate swelling reaction. The immediate reaction was followed by a sustained late reaction that peaked within 1 h and lasted for more than 6 h. Histologic examination of the footpads, obtained at hour 2, revealed that MCP-1 caused mild mononuclear cell infiltrates, moderate degranulation of mast cells, and soft tissue swelling. In contrast, MIP-1 alpha induced a severe inflammatory reaction that consisted of neutrophils, mononuclear cells, and degranulated mast cells. Electron microscope examination of the tissue revealed features of extensive mast cell degranulation by MIP-1 alpha and to a lesser extent by MCP-1. Thus, we conclude that mast cells are activated on injection of MCP-1, whereas degranulation of mast cells and recruitment of leukocytes contribute to the footpad reaction induced with MIP-1 alpha.
TNF-alpha binds to the N-terminal domain of fibronectin and augments the beta 1-integrin-mediated adhesion of CD4+ T lymphocytes to the glycoprotein.
Authors: Alon R , Cahalon L , Hershkoviz R , Elbaz D , Reizis B , Wallach D , Akiyama S K , Yamada K M , Lider O
Published: December 2022

Certain inflammatory cytokines and growth factors have been previously shown to interact with glycosaminoglycan moieties of the extracellular matrix (ECM). We have examined the association of the pleiotropic cytokine TNF-alpha with glycoprotein constituents of ECM. TNF-alpha interacted with fibronectin (FN) and laminin, and to a lesser degree with collagen. The major binding site for TNF-alpha on FN was localized to its 30-kDa N-terminal fragment (FN-N') with a Ki in the sub-nM range. The binding of 125I-labeled TNF-alpha to immobilized FN or FN-N' persisted for at least 24 h, and was specifically inhibited by antibodies to FN, mAb directed against the FN-N' domain, unlabeled TNF-alpha, and by the truncated forms of TNF-alpha receptors. Once bound to immobilized FN or FN-N', the cytokine could not be released by the soluble TNF-alpha-receptors, although it could be released by anti-TNF-alpha Ab. TNF-alpha was also found to interact with soluble FN, although with a lower affinity. Similar to the soluble cytokine, the FN-bound TNF-alpha appears to be functional; it augmented the beta 1-integrin-mediated adhesiveness of activated CD4+ human T cells to the glycoprotein. Hence, binding of TNF-alpha to immobilized FN, which modifies its functional accessibility to soluble TNF-alpha receptors, does not abolish but rather may locally restrict its activity. This study suggests that a major ECM glycoprotein can present, in a restricted manner, a functional adhesion-modulating cytokine to immune cells, and that ECM glycoproteins may regulate their intrinsic cell-adhesive properties by associating with cytokines.
Characteristics of a novel low affinity complement C3dg-binding protein of human platelets.
Authors: Chen H , Marjan J , Cox A D , Devine D V
Published: December 2022

The breakdown products of the complement protein C3 function in receptor-mediated immune clearance. The catabolism of the C3 molecule, mediated by factors I and H, results in the generation of the fragments C3c and C3dg. C3dg binds to human platelets in a specific and saturable manner. The direct interaction of platelets with soluble C3dg may contribute to immune-mediated platelet destruction. More importantly, platelets may interact with opsonized pathogens or complement-activating immune complexes via C3dg. In this report, we investigated the interaction of C3dg with platelets and calculated the Ka to be 3.2 x 10(6) M-1 with 1100 to 2200 specific binding sites/platelet. In the presence of 5 mM calcium, both the Ka and the number of specific binding sites were modestly decreased to Ka = 2.8 x 10(6) M-1 with 1400 to 2400 specific binding sites/platelet. The Scatchard plots demonstrated a curvilinear character. On labeling C3dg with peroxidase and visualizing platelet-bound C3dg by electron microscopy, it was shown that binding sites for C3dg were restricted to the platelet plasma membrane. Using a cell attachment assay, platelet adhesion to C3dg was readily detectable; attachment to C3dg-coated plates was not blocked by fibrinogen or fibronectin. We have characterized the C3dg-binding protein of platelets using the chemical cross-linkers, SASD and DSS, to cross-link C3dg to thrombin-stimulated platelets. Gel filtration of the 125I-labeled C3dg-platelet complex revealed the presence of a large protein complex that was absent when 125I-labeled C3dg alone was analyzed. SDS-PAGE of the radiolabeled cross-linked protein, followed by autoradiography, identified a 95-kDa membrane protein. The relationship of this C3dg-binding protein to other platelet membrane proteins has yet to be determined.
Inhibition of TNF by a TNF receptor immunoadhesin. Comparison to an anti-TNF monoclonal antibody.
Authors: Haak-Frendscho M , Marsters S A , Mordenti J , Brady S , Gillett N A , Chen S A , Ashkenazi A
Published: December 2022

TNF is an important mediator of inflammation, which can have deleterious effects when produced inappropriately. We have described a recombinant inhibitor of TNF, termed TNFR-IgG, or TNFR immunoadhesin, composed of the extracellular portion of the type 1 (p55) TNF receptor (TNFR) linked to the hinge and Fc regions of IgG heavy chain. This bivalent, Ab-like molecule is a potent inhibitor of TNF, exhibiting significantly higher affinity for the cytokine than soluble TNFR. Here, we compare the TNF-neutralizing capacity of TNFR-IgG to that of an anti-TNF mAb. In vitro, TNFR-IgG was 10- to 50-fold more potent than anti-TNF mAb at blocking the cytotoxic effect of exogenous TNF on actinomycin D-treated murine L-M cells. In vivo, the plasma half-life of TNFR-IgG in mice was approximately 6 days, similar to that reported for the anti-TNF mAb. However, the immunoadhesin was approximately 10-fold more effective than the Ab at neutralizing the activity of endogenous TNF, as assessed in a model for murine listeriosis. These results demonstrate a markedly greater potency of the TNFR immunoadhesin compared with the anti-TNF mAb at inhibiting TNF activity in vitro and in vivo.
Adherence of neutrophils induces release of soluble tumor necrosis factor receptor forms.
Authors: Lantz M , Björnberg F , Olsson I , Richter J
Published: December 2022

TNF is a potent activator of neutrophil granulocytes which acts via two cell surface receptors: the p55-TNF receptor (TNF-R55) and the p75-TNF receptor (TNF-R75). The extracellular region of the receptors can be released by proteolytic cleavage and form soluble TNF-binding proteins, TNF-R55-BP and TNF-R75-BP, respectively. The phorbol ester PMA, the chemotactic peptide FMLP, and TNF were all found to induce release of TNF-R55-BP and TNF-R75-BP from neutrophils in suspension in a time- and dose-dependent manner as measured by ELISA. Exposure of neutrophils to 10 ng/ml of PMA for 60 min resulted in release of 900 pg of TNF-R55-BP and 350 pg of TNF-R75-BP per 5 million cells, corresponding to approximately 4800 receptors per cell. In addition, adherence by itself of neutrophils to fibrinogen-coated culture plates and other surfaces resulted in a release of TNF-R55-BP of the same magnitude as seen in response of neutrophils in suspension to 1 nM TNF, whereas the release of TNF-R75-BP was less pronounced. The protein kinase C inhibitors staurosporin and calphostin C inhibited both the TNF-, PMA-, and adherence-induced release of soluble forms of TNFRs. Ab to the common beta-chain of the leukocyte integrins (CD18) did not affect adherence-induced TNF-R55-BP release, indicating that non-integrin-dependent mechanisms are involved in receptor cleavage. However, cross-linking of anti-CD18 Ab (IB4) with a Fab2 fragment resulted in a decrease of specific binding of 125I-TNF to neutrophils indicating that the leukocyte integrins can modulate TNFR expression on neutrophils. Thus, adherence to a biological surface, without additional stimuli, induces release of soluble TNFR form from neutrophils. TNFR expression can be modulated by protein kinase C as well as both leukocyte integrins and non-integrin-dependent adherence mechanisms.
Involvement of a botulinum toxin-sensitive 22-kDa G protein in stimulated exocytosis of human neutrophils.
Authors: Nath J , Powledge A , Wright D G
Published: December 2022

Studies of human peripheral blood neutrophils (PMNs) demonstrated that botulinum neurotoxin D (BT-D) ADP-ribosylates a 22-kDa PMN G protein (G22k) and inhibits the exocytosis of both specific and azurophilic granules stimulated by FMLP. Furthermore, this inhibition of PMN exocytosis by BT-D was found to be correlated with the degree of irreversible ADP-ribosylation of G22k by BT-D and to require modification of at least 85% of PMN G22k before significant inhibition of secretion is observed. Although both pertussis toxin and BT-D inhibited exocytosis in FMLP-stimulated PMNs, the inhibitory effects of the two toxins were found to be additive. Pertussis toxin and BT-D also inhibited Ca2+/GTP/GTP gamma S-induced secretion in digitonin-permeabilized PMNs, but there were distinct differences between the inhibitory effects of the two toxins. In contrast to BT-D, the exotoxin botulinum C3 was found to ADP-ribosylate primarily a 24- to 25-kDa PMN protein, and it was not found to inhibit Ca(2+)- and GTP-induced secretion in permeabilized PMNs. Ultrastructural studies of BT-D-treated PMNs showed an accumulation of distinct membrane-bound organelles in the periphery of the cells after FMLP stimulation, suggestive of a toxin-induced block in organelle-plasma membrane fusion. Taken together, these findings indicate that BT-D-sensitive G22k has a functional role in stimulated exocytosis of PMNs.
IFN and virus-inducible expression of an immediate early gene, crg-2/IP-10, and a delayed gene, I-A alpha in astrocytes and microglia.
Authors: Vanguri P , Farber J M
Published: December 2022

IFN-gamma is a potent macrophage activator and induces a number of early and delayed genes. crg-2, the presumed murine homologue of human IP-10, belongs to a family of proinflammatory chemokines and is induced as an immediate early gene in response to IFN-gamma in macrophages. In contrast, class II MHC or Ia genes which are essential for Ag presentation are induced as a delayed response to IFN-gamma. We studied the expression of crg-2 and compared it with Ia in astrocytes and microglia of the central nervous system since, like macrophages, these cells can also produce a number of cytokines, express Ia molecules, and present Ag. We showed that crg-2 mRNA was induced in astrocytes and microglia by IFN-gamma as well as a paramyxovirus, Newcastle disease virus (NDV). Crg-2 protein was detected in the cytoplasm and in the supernatants of IFN-gamma-treated astrocytes and microglia. IFN-gamma and NDV or UV irradiated-NDV (UV-NDV) also induced Ia mRNA in these cells. The kinetics of expression of crg-2 and Ia mRNA were compared in the same systems. While crg-2 mRNA appeared within 2 h and reached a maximum in 6 to 8 h, Ia mRNA was not detected before 8 h. Cycloheximide superinduced crg-2 mRNA induced by IFN-gamma or UV-NDV but it abolished Ia mRNA induction by the same stimuli. The induction of crg-2 in astrocytes and microglia likely contributes to the development of immune-mediated inflammation in response to viruses or in autoimmune diseases of the central nervous system.
Molecular analysis of IgA deficiency. Evidence for impaired switching to IgA.
Authors: Islam K B , Baskin B , Nilsson L , Hammarström L , Sideras P , Smith C I
Published: December 2022

The most common form of primary immunodeficiency is IgA deficiency (IgAD). However, the molecular basis of this disease remains elusive. Therefore, to address this issue we made a systematic analysis of the molecular events leading to IgA production. B lymphocytes that produce IgA have undergone somatic rearrangement that joins the switch (S) mu to S alpha region with deletion of the intervening sequences. Examination of the resulting S mu/S alpha junctions in unstimulated PBMC from IgAD patients by nested primer PCR revealed a significant decrease in the number of the S mu/S alpha fragments. To obtain the antisense primers to generate the S mu/S alpha fragments, we sequenced the human S alpha 1 and the downstream region extending to the C alpha 1 locus. Similar to previously reported switch sequences, we also found the S alpha 1 to be predominantly composed of pentameric repeats GAGCT and GGGCT. The decrease in the number of S mu/S alpha fragments is consistent with a profound decrease in the C alpha membrane mRNA expression in unstimulated PBMC, as well as in the C alpha mRNA levels and IgA production in PWM-stimulated PBMC. Sequence analysis of the switch junctions from IgA-producing cell lines, control donors, and an IgAD patient showed direct joining in 8 of 9 cases examined. TGF-beta 1, previously shown to be the switch factor for human and mouse IgA, was also examined. No difference in the TGF-beta 1 mRNA levels in unstimulated PBMC between the control subjects and the IgAD patients were detected. Our findings indicate that the failure to switch to IgA-producing B lymphocytes, or an impaired survival of such cells, may be an important molecular mechanism in IgAD.
B cells process and present lupus autoantigens that initiate autoimmune T cell responses.
Authors: Mamula M J , Fatenejad S , Craft J
Published: December 2022

Antibodies against U small nuclear ribonucleoprotein (snRNP) particles are a common finding in the sera of humans with SLE and in certain strains of mice with murine lupus. It is likely that Th cells are important in amplifying this autoantibody response. The focus of this work was to investigate events that might initiate autoimmune B and T cell response in non-autoimmune mice to native snRNP particles. Mice that were immunized and boosted with native mouse snRNPs failed to produce any detectable specific anti-snRNP antibody or T cell responses, suggesting that these autoreactive cells were deleted from the repertoire or were anergic to stimulation with this self Ag. In contrast, immunization with native foreign (human) snRNPs elicited both T cells and cross-reactive anti-snRNP antibodies; the latter predominantly were directed toward the A protein of the U1 snRNP. When mice were immunized with human and mouse snRNPs together in adjuvant, T cells specific for mouse snRNPs could be elicited. The results of these experiments suggested that the mechanism of breaking T cell tolerance to self snRNPs was dependent on the ability of cross-reactive B cells to process and present these autoantigens. To address this hypothesis, B cells purified from mice immunized with recombinant human A protein were transferred into naive mice. Upon boosting with native mouse snRNPs, autoreactive CD4+ T cells specific for mouse Ags, and not cross-reactive with human snRNPs, were observed. These studies support a model of molecular mimicry whereby autoantigen-presenting B cells are generated by foreign cross-reactive determinants that can, in turn, elicit an autoimmune T cell response.
T cell receptor alpha-chain repertoire of pathogenic autoantibody-inducing T cells in lupus mice.
Authors: Mao C , Osman G E , Adams S , Datta S K
Published: December 2022

The production of pathogenic anti-DNA autoantibodies in mice with lupus nephritis is dependent on special autoimmune Th cells that can also transfer the disease into preautoimmune mice. In previous work, these pathogenic Th cells were cloned and their TCR beta-chains were sequenced to reveal a recurrent motif of anionic residues in their CDR3 loops. Accordingly, approximately half of the Th clones were found to be specific for nucleosomal Ag that contain cationic residues. Herein, we analyzed the TCR alpha-chain repertoire of 15 of these pathogenic Th clones and found them to be heterogeneous, even among the nucleosome-specific Th clones. Most of these autoimmune TCR alpha-chains contained anionic residues in their CDR3 in addition to cationic residues. Therefore, these pathogenic Th clones of lupus probably recognize epitopes with mixed charge runs that are derived from autoantigens, such as histone-DNA complexes. Interestingly, the V alpha gene segments used by 10 of these Th clones derived from the (SWR x NZB)F1 lupus mice differed from previously reported sequences indicating that they were new members or alleles of the respective V alpha gene family. One of the Th clones used a gene from an entirely new murine V alpha gene family, identified here as V alpha 23, which consisted of approximately two members that were conserved among strains with different V alpha haplotypes. Knowledge of the primary structure of the TCR expressed by these pathogenic Th clones of lupus would help in the analysis of their antigenic specificities and also would be essential for studying their regulation in transgenic mice carrying these autoimmune TCR genes.
Variable regions of Ig heavy chain genes encoding antithyrotropin receptor antibodies of patients with Graves' disease.
Authors: Shin E K , Akamizu T , Matsuda F , Sugawa H , Fujikura J , Mori T , Honjo T
Published: December 2022

We have established EBV-transformed human B cell clones producing monoclonal antithyrotropin receptor antibodies from two patients with Graves' disease. We then isolated and characterized Ig H chain genes of 5 B cell clones with the thyrotropin-binding inhibitor Ig (TBII) activity and 4 B cell clones with the thyroid-stimulating antibody (TSAb) activity. We found that VH gene families used in the 5 TBII clones were all VH-III, although those of the four TSAb clones were diverse, including VH-II, -III, -IV, all -V. Most of VH segments used in TBII and TSAb are commonly used in other autoantibodies and fetal liver repertoire. The frequency of somatic mutations in TBII was higher than that in TSAb. Inasmuch as the same germline VH segment (V3-23) was used for both TBII and TSAb, the frequency and position of somatic mutations may be important for generation of TBII and TSAb.
In vitamin A deficiency multiple mechanisms establish a regulatory T helper cell imbalance with excess Th1 and insufficient Th2 function.
Authors: Cantorna M T , Nashold F E , Hayes C E
Published: December 2022

In hypovitaminosis A, Ab-mediated immunity is severely impaired. We reported that Trichinella spiralis infection stimulates a strong Th2 cell response in control mice but in vitamin A-deficient mice it stimulates a strong Th1 cell response. Here we investigated the immunobiologic mechanisms underlying this shift from a Th2- to a Th1-dominated response. A kinetic analysis showed that the Th1 cells developed first and IFN-gamma secretion predominated in deficient mice, whereas the Th2 cells developed later and IL-5 and IL-10 secretion predominated in control mice. The IFN-gamma-secreting cell frequencies were the same but cells from deficient mice secreted IFN-gamma sixfold faster than cells from control mice, and retinoic acid addition in vitro decreased that rate 50%. In contrast, the IL-5-secretion rates were the same but the IL-5-secreting cell frequency was lower in deficient mice than in controls, and retinoic acid addition in vitro doubled this frequency independently of its inhibitory effect on IFN-gamma. The APC from deficient mice stimulated greater IFN-gamma release than control APC and retinoic acid addition in vitro decreased this activity 50%. Together these results identify at least three vitamin A activities that balance Th1 and Th2 functions, down-regulating Th1 cell IFN-gamma secretion directly, decreasing activated APC function, and promoting Th2 cell growth and/or differentiation. In this system and perhaps others, the imbalance between regulatory Th1 and Th2 cells is one mechanism underlying poor Ab-mediated immunity in hypovitaminosis A.
Role of IL-6, IL-1, and CD28 signaling in responses of mouse CD4+ T cells to immobilized anti-TCR monoclonal antibody.
Authors: Holsti M A , McArthur J , Allison J P , Raulet D H
Published: December 2022

Purified CD4+ T cells require TCR engagement and Ag-nonspecific co-stimulatory signals to produce IL-2 and proliferate. A number of recent studies have demonstrated that the interaction of the B7 molecule expressed on APC with the T cell-associated CD28 molecule provides a potent co-stimulatory signal to both freshly isolated CD4+ T cells and cloned Th1 cells. Earlier reports have described the role of cytokines, in particular IL-6 and IL-1, as costimulatory molecules for T cell activation. We previously reported that IL-6 and IL-1 synergize to co-stimulate proliferation of purified mouse CD4+ T cells in conjunction with anti-TCR mAb. In this report we explore the interaction of IL-6, IL-1, and CD28 signaling in the activation of mouse CD4+ T cells, and demonstrate that the co-stimulatory requirements of the cells vary depending on the mode of TCR stimulation. CD28 signaling is not sufficient to co-stimulate responses of high buoyant density CD4+ T cells to anti-TCR-conjugated agarose beads; there is an additional requirement that can be supplied by exogenous IL-6 but not by IL-1. In contrast, in responses to anti-TCR mAb that is passively bound to the bottom of culture wells, CD28 stimulation is sufficient to co-stimulate proliferation, resulting in a very high level of IL-2 production; there is no additional requirement for exogenous IL-6 or IL-1. Possible explanations for the differential requirement for IL-6 in the two systems are discussed. Our results are consistent with the notion that CD28 signaling plays a central role in co-stimulating T cell responses. However, the results also suggest that, depending on the nature of the TCR stimulus, T cell activation may also require additional co-stimulatory signals provided by cytokines.
Role of DNA fragmentation in T cell activation-induced apoptosis in vitro and in vivo.
Authors: Mogil R J , Shi Y , Bissonnette R P , Bromley P , Yamaguchi I , Green D R
Published: December 2022

Apoptotic cell death, characterized by DNA fragmentation and morphologic changes, has previously been shown to occur in immature thymocytes and some T cell hybridomas after activation. Like some other forms of apoptosis, DNA fragmentation during activation-induced cell death precedes the morphologic events. For apoptosis to proceed, activation of the cells must persist at least to the time of DNA fragmentation, before which the cells can remain viable if the activation signal is removed. Aurintricarboxylic acid (ATA) blocks activation-induced apoptotic cell death in a T cell hybridoma, and kinetic studies show that this inhibition occurs at or near the time of DNA fragmentation in the cells. Taken together with the ability of ATA to inhibit DNA fragmentation in isolated nuclei exposed to Ca2+ and Mg2+, these data strongly suggest that ATA prevents apoptosis via its ability to inhibit endogenous endonuclease activity, and, conversely, that this activity is required for this form of cell death. In vivo, ATA inhibits thymocyte depletion and DNA fragmentation induced by anti-CD3 Ab. Further, specific loss of V beta 8+ thymocytes after administration of staphylococcal enterotoxin B is blocked by administration of ATA. These observations support an essential role for DNA fragmentation as an irreversible step in activation-induced apoptosis in T cell hybridomas and during T cell development. This is contrasted with heat shock-induced cell death, in which inhibition of DNA fragmentation does not prevent loss of cell viability.
In vitro adhesive interactions between rat lymphocytes and lacrimal gland acinar epithelium. Phenotype of adherent lymphocytes and involvement of adhesion molecules.
Authors: O'Sullivan N L , Skandera C A , Chin Y H , Montgomery P C
Published: December 2022

The selective interaction of trafficking lymphocytes with glandular epithelial cells is thought to link the lacrimal gland (LG) to the mucosal immune network. An in vitro binding assay was used to determine the phenotype of adherent cell populations and to study the involvement of lymphocyte adhesion molecules in the adherence process. Enriched B cell populations showed greater binding to LG epithelium than did enriched T cell populations. Direct phenotyping of adherent lymphocytes demonstrated that B lymphocytes (particularly IgA+ and IgG+ cells) were the predominant participants in LG binding. In vitro binding to LG acinar epithelium was inhibited by mAb with specificity for rat lymph node and Peyer's patch homing receptors and, to a lesser degree, by anti-VLA-4 and LFA-1 but not by anti-CD44 or Thy-1. In addition, the presence of rat lymph node and Peyer's patch homing receptors on endogenous LG lymphocytes was demonstrated by flow cytometry. These data show that B cells are the predominant population adhering to LG epithelium and suggest that lymphocyte homing receptors mediate the adherence process. These findings indicate that selective interactions between lymphocytes and the glandular epithelium contribute to the presence of IgA-producing cells in the LG.
Characterization of the human CD27 ligand, a novel member of the TNF gene family.
Authors: Hintzen R Q , Lens S M , Beckmann M P , Goodwin R G , Lynch D , van Lier R A
Published: December 2022

CD27 is a lymphocyte-specific member of the TNF/NGF-R family that is highly induced on T cells after TCR stimulation, primarily on lymphocytes belonging to the unprimed CD45RA+ subset. However, after prolonged activation both in vivo and in vitro, CD27 expression is gradually switched off. In search of physiologic signals that influence CD27 expression, we found that B cell lines can mediate down-regulation of CD27 surface expression on activated T cells via direct cell-to-cell contact and that preincubation of activated T cells with CD27 mAbs prevented this modulation. Based on these observations, we hypothesized that a ligand for CD27L is expressed on B cell lines and that ligand interaction would result in down-modulation of the molecule from the T cell surface. To identify this putative CD27L, mAbs were raised against the Burkitt cell line Ramos and screened for their ability to block down-modulation of CD27 on activated T cells. Using this approach, we isolated a mAb, designated 2F2, that inhibits CD27 down-regulation in a dose-dependent manner. We demonstrate that this mAb recognizes CD27L as it 1) reacted to mouse fibroblasts expressing the recently cloned CD27L, a novel surface-expressed member of the TNF gene family, and 2) prevented binding of a soluble CD27-Fc construct to a CD27L expressing cell line. Down-regulation of CD27 from the T cell surface by recombinant CD27L was shown to be at least partially caused by release of soluble CD27. The 2F2 mAb enabled us to begin to analyze the biochemical properties, tissue distribution, and function of the CD27L on human cells. From cell lysates of 125I surface-labeled Burkitt cells a complex immunoprecipitation pattern with dominant bands of 29, 55, and 122 to 127 kDa was obtained using the 2F2 mAb. Phenotypical analyses showed that freshly isolated lymphocytes lacked CD27L expression, but that expression of the molecule could be induced after in vitro stimulation of T and B cells. No expression was found on cells of the myeloid lineage or on endothelial cells. Finally, blocking of naturally expressed CD27L by the 2F2 mAb exerted a potent inhibitory effect on the proliferation of T cells in response to allogeneic B cells and PHA. The data presented in this paper identify CD27 and its ligand as potentially important structures involved in cellular interactions between T and B lymphocytes.