bluuebunny/crossref_metadata_2025 huggingface dataset:
Stem cell factor induces mast cell adhesion to fibronectin.
Authors: Dastych J , Metcalfe D D
Published: December 2022

Stem cell factor (SCF) or c-kit ligand is a growth factor cytokine produced by stromal cells that is known to influence mast cell proliferation and differentiation. We hypothesized that SCF may also influence the adhesion of mast cells to connective tissue matrix. To examine this hypothesis, we stimulated MCP5/L mast cells or murine bone marrow-derived cultured mast cells (BMCMC) with either SCF or PMA and observed adhesion to fibronectin (FN). As expected, 80 to 90% of PMA-activated MCP5/L cells or BMCMC adhered to FN. In addition, SCF promoted MCP5/L cell or BMCMC adhesion to FN in a dose-response fashion with 50 to 60% of BMCMC adhering to FN at a concentration 10 ng/ml of SCF. BMCMC adhesion was observed with as little as 200 pg/ml of SCF. Adhesion of SCF stimulated BMCMC to FN did not require IL-3, but was dependent on the concentration of FN used to coat the assay surface. Mast cell adhesion in the presence of SCF appeared to occur through an integrin receptor as adhesion was calcium dependent and could be blocked by an RGD (Ang, Gly, Asp)-containing peptide. SCF did not directly mediate adhesion through interaction with c-kit, as FN-coated surfaces exposed to SCF before initiation of the adhesion assay did not promote adhesion in the absence of soluble SCF. Rather, SCF appeared to stimulate adhesion to FN by activating mast cells through its interaction with c-kit. Thus, antibody to SCF blocked adhesion, and rat and murine SCF stimulated BMCMC adhesion to FN, but human SCF, which does not bind to murine c-kit, did not stimulate adhesion. Genistein, which inhibits tyrosine kinase activity, partially inhibited SCF-induced adhesion. SCF thus stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix under physiologic conditions.
Tyrosine phosphorylation provides an obligatory early signal for Fc gamma RII-mediated endocytosis in the monocytic cell line THP-1.
Authors: Ghazizadeh S , Fleit H B
Published: December 2022

The human monocytic cell line THP-1 expresses two classes of IgG Fc receptor (Fc gamma R), Fc gamma RI, a high affinity 72-kDa Fc gamma R, and Fc gamma RII, a low affinity 40-kDa Fc gamma R. Biochemical as well as indirect immunofluorescence studies demonstrated that the selective cross-linking of Fc gamma RII with either anti-Fc gamma RII mAb Fab followed by F(ab)2 fragments of goat anti-mouse IgG, or aggregated hIgG1, which represents a physiologic ligand for this receptor, resulted in the activation of a protein tyrosine kinase (PTK). Several distinct cellular proteins including the Fc gamma RII itself were specifically phosphorylated on tyrosine upon ligand binding. Cross-linking of Fc gamma RII also triggered a rapid internalization of Fc gamma RII that was dependent upon tyrosine kinase activity. The internalization of the receptor in endocytic vesicles was established by confocal microscopy. The time course of Fc gamma RII-initiated tyrosine phosphorylation paralleled endocytic events and reached a maximum between 5 and 10 min after ligand binding and declined toward basal levels as endocytosis was completed. Identical concentrations of genistein, an inhibitor of PTK, blocked Fc gamma RII-mediated endocytosis as well as the induction of tyrosine phosphorylation of Fc gamma RII and other cellular proteins. Cross-linking of Fc gamma RI also induced a rapid tyrosine phosphorylation of cellular proteins similar to the Fc gamma RII-mediated events. However, Fc gamma RII was not tyrosyl phosphorylated upon Fc gamma RI activation. Thus Fc gamma RII is a unique substrate for the PTK activity associated with Fc gamma RII upon cross-linking of this receptor. These results support the conclusion that Fc gamma RII is capable of independent signaling on monocytic cells and that protein tyrosine phosphorylation is an obligatory proximal signal for Fc gamma RII-mediated endocytosis. Furthermore, the signaling pathways employed by Fc gamma RI and Fc gamma RII are likely to be distinct.
Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status.
Authors: Gorski J , Yassai M , Zhu X , Kissela B , Kissella B [corrected to Kissela B ] , Keever C , Flomenberg N
Published: December 2022

The analysis of the T cell repertoires involved in local or systemic immune responses is beginning to play an important role in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, alloimmunity including allograft rejection, and tumor immunity. Here we analyze circulating T cell repertoires by determining TCR beta-chain gene complexity using a modification of V beta family-specific PCR. This approach, called CDR3 size spectratyping, uses the size heterogeneity of the CDR3 as a further source of specificity in TCR analysis. It has been used here to analyze the complexity and stability of circulating T cell repertoires in normal adults, including bone marrow donors, and bone marrow transplant recipients. Normal spectratypes are both complex and stable. The repertoire complexity of marrow recipients correlates with their state of immune function. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. Spectratype analysis is applicable to other studies of specific repertoire skewing such as may be associated with immunodeficiency or found at sites of immune activity.
Costimulation through CD28 enhances T cell-dependent B cell activation via CD40-CD40L interaction.
Authors: Klaus S J , Pinchuk L M , Ochs H D , Law C L , Fanslow W C , Armitage R J , Clark E A
Published: December 2022

Changes in T cell helper function were analyzed when anti-CD3-activated T cells were costimulated with mAbs to the CD28 receptor (anti-CD28). T cell-dependent B cell growth and differentiation were consistently augmented if anti-CD3 stimulated-T cells were simultaneously activated with anti-CD28. Although anti-CD28 enhanced IL-2 and IL-4 production, it did not increase B cell responses solely by augmenting production of soluble lymphokines. Anti-CD28 costimulation induced increases on T cells of CD40 ligand (CD40L), known to promote B cell proliferation and Ig secretion. Because anti-CD28 promoted T cell helper functions and expression of CD40L, we examined the dependence for CD40L during T cell-dependent B cell responses. Although soluble CD40 fusion proteins only partially inhibited T cell-dependent B cell activation, we found a strict requirement for CD40L expression at initiating B cell responses. Both CD40L expression and T cell help were blocked by cyclosporin A after TCR cross-linking, and, unlike T cell proliferation, both remained cyclosporin A sensitive during CD28 costimulation. In addition, anti-CD28 could not compensate for the T cell helper deficiency of hyper IgM syndrome patients who lack functional CD40L. Thus, anti-CD28-induced T cell help is delivered via a CD40L-dependent process. The fact that cross-linking CD40 on B cells promotes expression of the B7/BB-1 ligand for CD28 suggest T and B interactions may have a reciprocal amplification mechanism.
Tissue-specific expression of messenger RNAs encoding endogenous viral superantigens.
Authors: Jarvis C D , Germain R N , Hager G L , Damschroder M , Matis L A
Published: December 2022

The minor lymphocyte stimulating (MLS) superantigens of mice are encoded by open reading frames (ORFs) in the 3' long terminal repeats (LTRs) of endogenous mouse mammary tumor viruses. By stimulating all T cells bearing particular TCR V beta proteins, these viral superantigens (v-SAGs) exert profound effects on T cell development and function. We have examined expression of the 1.7 kb mRNA product predicted to encode v-SAG proteins in different cells and tissues of the immune system. The LTR-ORF mRNA was expressed in activated B cells and activated mature CD8 but not CD4 T cells, consistent with previous functional studies assessing MLS activity in these cell types. Little or no message was detected in thymic epithelial cells, macrophages, or dendritic cells, although low levels could be observed in thymic epithelium after southern hybridization to PCR-amplified cDNA. LTR-ORF mRNA was also expressed in immature CD4-CD8- and CD4+CD8+ thymocytes, suggesting selective down-regulation of expression in the T cell lineage after differentiation to the CD4+ phenotype. Thus, among cells of the immune system, v-SAG encoding mRNA is expressed predominantly within the lymphoid lineage.
Unique phenotype and distinct TCR V beta repertoire in human peripheral blood alpha beta TCR+, CD4-, and CD8- double negative T cells.
Authors: Niehues T , Gulwani-Akolkar B , Akolkar P N , Tax W , Silver J
Published: December 2022

TCR-alpha beta+ CD4- CD8- double-negative (DN) T cells represent a small, poorly defined T cell subset in human peripheral blood that has been postulated to be potentially autoreactive. To define some of the characteristics of this subset of T cells, DN cells and CD4+ and CD8+ single-positive (SP) cells were purified from the peripheral blood of six unrelated individuals by a combination of positive selection and depletion using mAb conjugated to immunomagnetic beads. Purified DN cells were found to be enriched in cells expressing HLA-DR and the NK cell marker, CD56, when compared to the SP population. Furthermore, in contrast to SP cells that express the adhesion marker CD44, DN cells were found to express very little, if any, CD44. When the V beta TCR repertoires of DN and SP (CD4+ and CD8+) cells, determined by quantitative (q) PCR, were compared all three populations were found to be considerably different. Furthermore, several V beta segments (V beta 11 and V beta 19) were consistently expressed at higher levels on DN cells than on SP cells. The TCR repertoires of both DN and SP cells were frequently characterized by dominance of one or more V beta segments that could in some instances be shown to be restricted to the CD45RO+ ("memory") population. However, differences in TCR repertoire between DN and SP cells were observed even when CD45RO+ cells were removed before qPCR analysis. These studies suggest that the TCR repertoires of DN and SP cells are determined by different selection mechanisms and that DN and SP cells are directed against different Ag.
Influence of coding region polymorphism on the peripheral expression of a human TCR V beta gene.
Authors: Vissinga C S , Charmley P , Concannon P
Published: December 2022

A number of human TCR V beta gene segments are reported to be polymorphic, with alleles differing by one or a small number of amino acid substitutions. In the absence of detailed structural information regarding the interaction of specific positions in the TCR with Ag or MHC, the significance of such variation is difficult to assess. In this report the relative use of the two common alleles of the human V beta 6.7 gene, 6.7a and 6.7b, which differ by two non-conservative amino acid substitutions, and the use of two common alleles of the V beta 12.2 gene, which differ by only silent substitutions, were measured in PBL derived from individuals heterozygous for these alleles. Equal use of V beta 12.2 alleles was observed, consistent with the inability of selection mechanisms to discriminate between the products of these alleles that are indistinguishable at the amino acid level. However, statistically significant skewing in the use of V beta 6.7 alleles was observed in 15 of 16 individuals studied. Expression levels for each allele ranged from 16 to 84% of the total V beta 6.7 signal in heterozygous individuals, with either the 6.7a or the 6.7b allele predominant in different individuals. Based on segregation studies in families, it seems unlikely that other unidentified polymorphism in the TCR beta locus, such as in the V beta 6.7 promoter, was responsible for the differential allele expression. Family studies provided no evidence for an association between specific HLA haplotypes and V beta 6.7 allele use. These results indicate that even modest allelic variation in human TCR V beta coding regions can have a significant impact on the expression of human V beta genes in the peripheral repertoire.
T cell receptor recognition of MHC class II alloantigens is highly cell type dependent.
Authors: Katz J F , Sant A J
Published: December 2022

According to some models of T cell education, tolerance, and autoimmunity, recognition of MHC molecules by T cells may depend on the nature of the APC expressing the MHC/Ag complex. To examine this, a panel of 23 I-Ad-restricted, alloreactive T cells were used to probe MHC class II molecules expressed on established lines representing different lineages. Surprisingly, we observed cell type-specific reactivity in the majority of the T cells. In all, 18 different reactivity patterns were identified. The patterns observed suggests that MHC reactivity can be organized into a hierarchical pattern for both the APC and the T cells. Experiments assessing T cells' avidities for allogeneic targets, ability to produce lymphokines, and expression of accessory molecules revealed no predictive correlation with the hierarchical reactivity pattern, nor did experiments measuring allogeneic target cells' expression of known accessory molecules and ability to stimulate Ag-specific hybridomas. These results suggest that the differential reactivity cannot be accounted for by accessory molecule discrepancies among the APC, but rather might reflect deficiencies in the ability of the various APC to engage the Ag-specific T cell receptor. Collectively, these data indicate that a significant fraction of allorecognition of MHC class II is cell-type dependent and that cell-type-specific recognition may relate to peptide-specific recognition requirements by the Ag receptor of alloreactive T cells.
Tyrosine kinase-dependent activation of human NK cell functions upon stimulation through a 58-kDa surface antigen selectively expressed on discrete subsets of NK cells and T lymphocytes.
Authors: Melero I , Salmerón A , Balboa M A , Aramburu J , López-Botet M
Published: December 2022

We have raised a mAb, termed HP-3E4 (IgM), by immunizing BALB/c mice against human IL2-activated NK cells. The HP-3E4 mAb recognized in different donors variable proportions (< 2-70%) of either fresh or activated NK cells. A small population of T cells (alpha/beta and gamma/delta) appeared HP-3E4+ in PBL. No reactivity was detected on other leukocytes and a panel of cell lines from different lineages. By immunohistochemical staining of different tissues, few HP-3E4+ cells were detected only in lymphoid organs. Analysis of CD56+CD16+CD3- clones (n = 167) from unrelated donors (n = 6), showed that the Ag was stably expressed on 8 to 70%, moreover it was detected on some gamma/delta + T cell clones, whereas all CD3+ alpha/beta +(CD4+ and CD8+) clones analyzed (n = 90) were HP-3E4-. As assessed by SDS-PAGE analysis, the HP-3E4 mAb immunoprecipitated a 58-kDa surface structure. When compared with two mAbs (GL183 and EB6) previously reported to bind also a clonally distributed 54- to 58-kDa Ag, the HP-3E4 mAb appeared to recognize a distinct epitope, thus allowing to further define NK cell subsets. Stimulation of IL2-activated NK cells with the mAb triggered TNF-alpha and IFN-gamma production, which was enhanced by using the mAb attached to plastic or in the presence of suboptimal concentrations of phorbol esters. Although the HP-3E4 mAb did not significantly modify NK cell-mediated cytotoxicity against different targets, with the exception of the Hmy-C1R cell line, it activated BLT esterase secretion. Remarkably, the HP-3E4 mAb triggered phosphoinositide turnover and an early increase of [Ca2+]i, as well as tyrosine phosphorylation of several cellular substrates including CD3 zeta; inhibition of tyrosine kinase activity with genistein hampered the HP-3E4-mediated stimulation of cytokine production. Our data provide further support for the structural diversity of a 58-kDa surface Ag, whose expression is restricted to discrete NK and T cell subsets. Moreover, the results support the fact that the molecule plays an active role in regulating NK cell functions through signal transduction mechanisms comparable to those triggered via Fc gamma RIII.
V beta 5+ T cell receptors skew toward OVA+H-2Kb recognition.
Authors: Dillon S R , Jameson S C , Fink P J
Published: December 2022

T cells recognize a complex of peptide Ag bound within the groove of MHC-encoded molecules. Although many studies have attempted to correlate TCR gene expression with specificity for particular Ag/MHC combinations, it is still not clear exactly how the TCR physically interacts with its cognate ligand. We have analyzed transgenic mice that carry a rearranged gene encoding a V beta 5.2+ TCR beta-chain derived from the CD8+ CTL clone B3, which is specific for chicken OVA+H-2Kb. Surprisingly, we have found that peripheral lymphocytes isolated from naïve V beta 5.2 transgenic mice can generate a strong primary anti-OVA CTL response when stimulated in vitro with OVA+H-2b, whereas generation of even a weak anti-OVA response from nontransgenic littermates requires in vivo priming. This response is Ag specific, because the transgenic mice are unable to respond with or without priming to vesicular stomatitis virus, which contains a dominant epitope presented in the context of H-2Kb. The precursor frequency of OVA-specific CTL in unprimed V beta 5.2 transgenic mice is approximately 30-fold higher than that in nontransgenic littermate controls. Reverse transcription-PCR analyses demonstrate that OVA-specific CTL lines derived from unprimed V beta 5.2 transgenic mice express a variety of TCR V alpha elements, indicating that the transgenic anti-OVA response is not solely due to the reconstitution of the original B3 TCR. In fact, our data suggest that even a nontransgenic V beta 5+ TCR is intrinsically OVA specific. First, five separate OVA-specific oligoclonal CTL lines derived from individual nontransgenic mice demonstrate dramatic skewing toward expression of V beta 5.1+ or V beta 5.2+ TCR over the course of several in vitro stimulations. Second, sorting for V beta 5+CD8+ nontransgenic cells enriches for OVA-specific CTL. However, peptide antagonism experiments using mutant forms of the Kb-restricted OVA peptide reveal distinct differences between the recognition patterns of two individual OVA-specific CTL lines derived from unprimed V beta 5.2 transgenic mice. These experiments support the notion that a discrete portion of the responding TCR can heavily influence but not necessarily be solely sufficient for the recognition of a peptide Ag presented in the cleft of an MHC-encoded molecule.
Monocytes use either CD11/CD18 or VLA-4 to migrate across human endothelium in vitro.
Authors: Meerschaert J , Furie M B
Published: December 2022

Monocytes traverse the endothelial lining of blood vessels and migrate into both normal and inflamed tissues. An in vitro model of a vascular wall, consisting of HUVEC cultured on acellular human amniotic tissue, was employed to examine the roles of several adhesion molecules in diapedesis of monocytes. Approximately half of the monocytes added to this system traversed the endothelium in a time-dependent fashion, completing their migration within 2 h. Pretreatment of HUVEC with IL-1 beta for 4 h increased the rate of adhesion of monocytes, but did not alter the number that ultimately migrated. A mAb to CD18, ts1/18, greatly inhibited adhesion and migration of monocytes when the monocytes were incubated with unstimulated HUVEC monolayers for 20 min. Much less inhibition was observed when the incubation period was increased to 2 h or when HUVEC were pretreated with IL-1 beta. A mAb to VLA-4, HP1/2, had little or no inhibitory effect in all cases. The combination of ts1/18 and HP1/2 greatly inhibited (up to 98%) adhesion and migration of monocytes across both unstimulated and IL-1 beta-stimulated monolayers. Additional inhibition experiments indicated that VLA-4 interacted with unstimulated endothelium by binding to VCAM-1 and, to a much lesser extent, fibronectin. These results suggest that monocytes are capable of interacting with endothelium during diapedesis via either CD11/CD18- or VLA-4-dependent pathways.
A synthetic peptide from complement protein C9 binds to CD59 and enhances lysis of human erythrocytes by C5b-9.
Authors: Tomlinson S , Whitlow M B , Nussenzweig V
Published: December 2022

The membrane glycoprotein CD59 protects host cells from homologous complement attack by inhibiting the assembly of the membrane attack complex. CD59 binds to C8 and C9 in the nascent membrane attack complex and interferes with C9 membrane insertion and polymerization. We show here that a synthetic peptide from the putative C9 hinge region, postulated to be involved in the rearrangement of C9 globular domains during membrane insertion, binds specifically to CD59 and enhances lysis of human erythrocytes by the terminal complement C5b-9 complex. The peptide, C9H, caused a dose-dependent increase in the sensitivity of human erythrocytes to C5b-9-mediated lysis by interfering with the final C9 binding and/or membrane insertion step. C9H exhibited species-specificity, since it had no activity against guinea pig C8 and C9 or on the putative functional homologues of CD59 in guinea pig erythrocytes. A direct association between CD59 and C9H was suggested by two different binding experiments: C9H inhibited the binding of 125I-labeled CD59 to immobilized C9, and C9H immobilized to microtiter plates bound purified CD59 and selectively recognized CD59 from extracts of detergent-solubilized human erythrocyte membranes. These data indicate that the peptide C9H corresponds to a region of the CD59 binding site of C9.
Monoclonal antibody 2D10 recognizes a novel T cell costimulatory molecule on activated murine B lymphocytes.
Authors: Chen C , Faherty D A , Gault A , Connaughton S E , Powers G D , Godfrey D I , Nabavi N
Published: December 2022

We have developed a panel of rat mAbs against dibutyryl cAMP-activated 5C2 cells. In this panel, one mAb, 1G10, recognized murine B7. Another mAb designated 2D10 did not bind to murine B7 but could recognize a surface molecule expressed only on dibutyryl cAMP-activated 5C2 mouse B lymphoma cells or on LPS-stimulated splenic B cells. This new molecule is referred to as early T cell costimulatory molecule-1 (ETC-1). From both activated 5C2 cells and splenic B cells, mAb 2D10 immunoprecipitated a 59- to 60-kDa protein, which was different from the 47- to 55-kDa murine B7 protein precipitated from the same cell populations. FACS analysis showed that, in contrast to B7, the expression of ETC-1 on 5C2 cells was induced by lower concentrations of dibutyryl cAMP and displayed a faster kinetics. LPS-stimulated splenic B cells expressed relatively low levels of B7 and much higher levels of ETC-1. Importantly, in an Ag presentation assay using activated 5C2 cells as APC, the secretion of IL-2 by C8A3 T hybrids was partially inhibited by mAb 2D10 alone and completely blocked by combination use of mAbs 2D10 and 1G10 in a dose-dependent and synergistic fashion. In a one-way primary MLR, mAb 2D10 alone at 0.1 to 1 microgram/ml inhibited T cell proliferation by 19 to 56%. However, an additive blocking effect (up to 76%) was observed when two mAbs were added in combination. Thus, our data have demonstrated that a novel T cell costimulatory molecule is present on activated murine B cells, which, in cooperation with B7, may play a critical role in optimal T cell activation.
Isolation and characterization of a human nonspecific suppressor factor from ascitic fluid of systemic lupus erythematosus. Evidence for a human counterpart of the monoclonal nonspecific suppressor factor and relationship to the T cell receptor alpha-chain.
Authors: Xavier R M , Nakamura M , Tsunematsu T
Published: December 2022

The monoclonal nonspecific suppressor factor (MNSF) is a lymphokine produced by a murine T cell hybridoma capable of suppressing Ab production by LPS-stimulated B cells. The existence of a human counterpart of MNSF, designated as the human nonspecific suppressor factor (hNSF), was likely because the anti-MNSF mAb (MO6) recognizes a similar suppressive activity in supernatants of Con A-stimulated human PBMC. By using the MO6 mAb, we investigated the presence of hNSF in the ascitic fluid of a patient with SLE. A small amount of cross-reactive hNSF was isolated from concentrated ascitic fluid fractionated with the MO6-affinity column, and a specific anti-hNSF mAb (P2) was produced. The hNSF eluted from the P2-affinity column could suppress up to 80% of the PWM-induced IgG production of human PBMC in a dose-dependent manner, even when added in late culture periods. Moreover, hNSF could inhibit proliferation of PBMC triggered by either PWM or Con A, which also implies an effect on T cells. On SDS-PAGE, the isolated hNSF resolved as a single peak of about 66 kDa and probably represents an aggregate of hydrophobic subunits. On reverse-phase HPLC, the bioactivity could be recovered from a single peak at 18.3 min. The suppression of IgG production induced by hNSF could be partly neutralized by preincubation with an anti-TCR-alpha mAb, whereas an anti-TCR-beta did not have any effect. Anti-TCR-alpha could also directly bind to the isolated nNSF, demonstrating some serologic relationship, as has been reported for several Ag-specific suppressor systems.
Neutralization of IL-8 inhibits neutrophil influx in a rabbit model of endotoxin-induced pleurisy.
Authors: Broaddus V C , Boylan A M , Hoeffel J M , Kim K J , Sadick M , Chuntharapai A , Hébert C A
Published: December 2022

Although the potent neutrophil chemotaxin, IL-8, is a known product of endotoxin-stimulated cells in vitro, the contribution of IL-8 to neutrophil recruitment in Gram-negative endotoxin inflammation in vivo is unknown. To determine whether neutralization of IL-8 would decrease endotoxin-induced neutrophil influx, we generated neutralizing mAbs to rabbit rIL-8 for use in our rabbit model of endotoxin-induced pleurisy. One mAb, ARIL8.2, specifically inhibited both rabbit rIL-8-induced chemotactic activity and activation of the rabbit IL-8 receptor transfected in 293 cells. Anesthetized rabbits with in-dwelling pleural catheters received either neutralizing mAb (ARIL8.2; 1 mg/kg) or irrelevant isotype-matched mAb (anti-HIV gp120) i.v. 1 h before as well as intrapleurally (20 micrograms/ml) at the time of intrapleural instillation of Escherichia coli endotoxin (200 ng bilaterally). ARIL8.2 blocked 77% of endotoxin-induced neutrophil influx (21 +/- 2 (SE) x 10(6) (ARIL8.2) vs 91 +/- 15 x 10(6) (anti-gp120) (p < 0.0001)). By Western analysis, a band corresponding to rabbit IL-8 was detected in the pleural liquid of rabbits in both groups. By ELISA, however, the concentration of free, unbound IL-8 in the pleural liquid was significantly less in the ARIL8.2 group than in the anti-gp120 group for at least 4 h, confirming that ARIL8.2 bound the IL-8 generated in vivo during that time. We conclude that neutralization of IL-8 profoundly inhibits neutrophil recruitment in endotoxin-induced pleurisy indicating that IL-8 is a major chemotactic factor in this model of acute inflammation.
Regulation of neutrophil-derived chemokine expression by IL-10.
Authors: Kasama T , Strieter R M , Lukacs N W , Burdick M D , Kunkel S L
Published: December 2022

In this study, we examined IL-10 regulation of polymorphonuclear leukocyte (PMN)-derived chemokine expression. Studies demonstrated that IL-10 dose dependently suppressed the expression and production of PMN-derived macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, IL-8 mRNA, and protein. Although inhibition of protein synthesis was found to superinduce the expression of PMN-derived chemokine steady-state mRNA, the inhibitory activity of IL-10 was completely abrogated in the presence of either cycloheximide or puromycin. These data suggest that the effect of IL-10 on PMN-derived chemokine expression was through the production of de novo repressor protein(s). Next, we examined the half-life (t1/2) of chemokine mRNA by LPS-treated PMNs in the presence or absence of IL-10. The t1/2 of MIP-1 alpha, MIP-1 beta, and IL-8 mRNA from PMNs treated for 4 h with LPS before actinomycin-D (Ac-D) addition were approximately 40 min, 1.7 h, and 2 h, respectively, whereas the t1/2 from PMNs stimulated for 8 h before Ac-D were 2, 2, and > 9 h, respectively. Interestingly, IL-10 significantly accelerated the decay of all three of the above chemokine mRNA. The t1/2 of MIP-1 alpha, MIP-1 beta, and IL-8 mRNA from PMNs treated with LPS plus IL-10 compared with LPS alone was reduced by 62, 50, and 40%, respectively, at the 4-h time point and by 50, 25, and 70%, respectively, at the 8-h time point. These findings support the notion that PMNs are an important cellular source of both C-X-C and C-C chemokines, and that IL-10 regulates both inflammatory/immune responses by not only modulating the activities of T cell, B cell, and mononuclear phagocyte function, but also by inhibiting PMN-derived chemokine expression.
Molecular characterization of human Ig heavy chain DIR genes.
Authors: Sanz I , Wang S S , Meneses G , Fischbach M
Published: December 2022

Antibody VDJ recombination is ensured by evolutionarily conserved recombination signals (RS). The 12/23 rule postulates that only gene segments with asymmetrically spaced RS recombine with one another. Two unusually long D genes (170 bp) with irregular RS (DIR) have been reported in humans and have been postulated to participate actively in VDJ recombination, thus frequently contributing to the Ab heavy chain third hypervariable region (CDR3). However, the limited sequence information retained in the CDR3 along with significant sequence diversity has precluded an accurate assessment of the actual role and genomic diversity of DIR genes. Furthermore, DIR genes pose an interesting puzzle in terms of their precise mechanism of recombination because they possess multiple and imperfect RS, often located up to 30 bp away from the recombining fragment within the DIR coding region. Here we present conclusive evidence for the existence of additional human germ-line DIR genes and preliminary evidence that suggests the absence of DIR-like sequences in nonprimate animals. We also show that DIR genes are under the transcriptional control of VH-independent promoters and have the potential to encode a D mu protein of 105 amino acids. Finally, DIR genes seem at least in early fetal life to recombine preferentially through the conventional 3' RS.
Slow release of soluble TNF receptors by monocytes in vitro.
Authors: Leeuwenberg J F , Jeunhomme T M , Buurman W A
Published: December 2022

In this study, we investigated the release of soluble(s) TNF-R by PBMC in vitro. T cell activation by mAb anti-CD3, as well as activation with phorbol esters (PMA), enhanced the release of both sTNF-R55 and sTNF-R75 by PBMC. In contrast to shedding of TNF-R by neutrophils upon activation, release of sTNF-R by PBMC proved to be a relatively slow process, reaching a plateau after 2 days of culture. Monocytes appeared to be the main source of the released sTNF-R, whereas activation of purified T cells induced only a minor release of sTNF-R as compared with the whole cell population. To unravel the mechanism, a number of cytokines were added during a 2-day culture of cells. IL-10 enhanced sTNF-R levels with similar kinetics as mAb anti-CD3 and PMA, whereas the other cytokines tested did not affect the release of sTNF-R by PBMC, pure T lymphocytes, or purified monocytes, either activated or not. Conversely, inhibitors of cytokines were added during the activation period to study the effect of endogenously produced cytokines on sTNF-R release. mAb anti-IL-10 and IL-1ra partly sTNF-R release, whereas other inhibitors did not affect the release. The results obtained in vitro may extend our insight in the mechanism via which sTNF-R are enhanced in vivo during inflammatory reactions.
Pertussis toxin interferes with superantigen-induced deletion of peripheral T cells without affecting T cell activation in vivo. Inhibition of deletion and associated programmed cell death depends on ADP-ribosyltransferase activity.
Authors: Gonzalo J A , González-García A , Baixeras E , Zamzami N , Tarazona R , Rappuoli R , Martínez C , Kroemer G , Tarazona R , Terezone R
Published: December 2022

Intravenous injection of a bacterial superantigen such as Staphylococcus aureus enterotoxin B (SEB) causes transient activation and expansion of SEB-reactive V beta 8+ T cells, as well as specific down-regulation of the immune response, through partial deletion of superantigen-reactive T cells. Here we demonstrate that co-administration of pertussis toxin (PTX) and SEB reduces the SEB-induced deletion of V beta 8+ T cells, although it does not affect T cell activation and proliferation. PTX abrogates the SEB-driven deletion of V beta 8+CD4+ (not V beta 8+CD8+) splenocytes that is observed early (12-24 h) after SEB injection. Moreover, it antagonizes the late (> or = 4 days) deletion of V beta 8+CD4+ and V beta 8+CD8+ peripheral T cells that follows transient expansion of such cells. This phenomenon is associated with significant reductions in apoptosis and endonucleolysis and is not caused by a compensatory increase in proliferation of SEB-reactive T cells, as we determined by using a combined fluorometric analysis of cell cycle and DNA alterations, which are associated with programmed cell death. These effects are also observed in thymectomized animals, thus excluding the possibility that PTX might act by enhancing the maturation and export of thymic T cells to the periphery. Moreover, the SEB-induced reduction of V beta 8+ splenocytes is antagonized by PTX in vitro. The capacity of PTX to reduce clonal deletion depends critically on its ADP-ribosyltransferase activity, inasmuch as a non-enzymatic PTX mutant fails to act in this biologic system. We conclude that PTX selectively antagonizes or impedes the delivery of negative signals to T cells, which are stimulated by superantigens, without interfering with the transmission of stimulatory signals.
Inhibition of in vivo tumor growth by the beta chemokine, TCA3.
Authors: Laning J , Kawasaki H , Tanaka E , Luo Y , Dorf M E
Published: December 2022

TCA3 is a proinflammatory murine glycoprotein that shares structural features with cytokines of the beta chemokine family and is a chemoattractant for neutrophils and monocytes. To assess the in vivo functions of TCA3, the cDNA was expressed in two mouse myeloma cell lines. Although the transfected and control cells had similar growth rates in vitro, TCA3-expressing tumors demonstrated impaired growth in both normal and immunodeficient mice. Histologic evaluation of the injection sites demonstrated that TCA3 expression resulted in an early neutrophil and monocyte infiltrate accompanied by tumor necrosis. There was complete regression of the TCA3-transfected tumor in some immunocompetent syngeneic mice. The TCA3-transfected cells induced specific and long-lasting immunity in mice that showed complete tumor regression; these animals were resistant to challenge with nontransfected tumor cells. In contrast, priming with irradiated tumor cells provided little protection against challenge with nontransfected tumor, which indicates that TCA3 specifically augments tumor immunogenicity. Mixing TCA3-transfected cells with normal tumor cells causes retarded growth of the normal tumor cells provided the latter are injected into the same site. Furthermore, direct in situ injection of soluble rTCA3 early during the course of tumor implantation also inhibits tumor growth. The data suggest that TCA3 may perform two roles in tumor protection: it induces lymphocyte-independent antitumor activity and stimulates tumor-specific immunity. We speculate that TCA3 has natural adjuvant activities that result in augmented immune responses.
Modulation of the in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. III. Enhancement by RP-3 treatment of the anti-sheep red blood cell plaque-forming cell response in rats.
Authors: Tamura M , Sekiya S , Terashita M , Sendo F
Published: December 2022

Because recent work has shown that neutrophils produce various cytokines and are activated by these agents, this study was undertaken to examine neutrophil participation in immune networks. In our previous work, we demonstrated that delayed type hypersensitivity to SRBC and accompanying mononuclear leukocyte recruitment are inhibited in vivo by selective depletion of neutrophils using a mAb designated RP-3. To elucidate the function of these cells in Ab production, we assessed the splenic plaque-forming cell response to SRBC in a rat model through selective depletion of circulating neutrophils by RP-3. This depletion which occurred at the time of immunization resulted in an increase in the number of anti-SRBC Ab producing cells, as determined by the direct and indirect plaque-forming cell response. This phenomenon was observed only when the Ag was administered i.p. and not with i.v. immunization. These results suggest that neutrophils suppress Ab production in certain situations.
Xanthine dehydrogenase and xanthine oxidase activity and gene expression in renal epithelial cells. Cytokine and steroid regulation.
Authors: Pfeffer K D , Huecksteadt T P , Hoidal J R
Published: December 2022

Reactive oxygen species have been implicated in the tissue injury and loss of epithelial barrier function associated with a number of clinical disorders in which disregulated inflammation seems to be a dominant event, such as endotoxemia and viral syndromes. In these disorders, xanthine oxidase (XO) contained within the epithelial cell has been proposed as a major source of injurious reactive oxygen species. This study was undertaken in an effort to understand the regulation of xanthine dehydrogenase (XDH)/XO expression at both the activity and gene expression levels in the epithelial cell under conditions associated with the inflammatory response. The results indicate that TNF, IFN-gamma, IL-6, IL-1, and dexamethasone induce XDH/XO activity in bovine renal epithelial cells (MDBK). This pattern of XDH/XO regulation by cytokines and steroids is analogous to the profile of response seen by acute phase reactants. Metabolic labeling and immunoprecipitation revealed the increase in XDH/XO activity requires new protein synthesis. By Northern analysis, all cytokines and dexamethasone increased the level of the 5-kb XDH/XO mRNA. This increase was not detectable in the presence of actinomycin D but was further induced in the presence of cycloheximide, consistent with the major site of XDH/XO up-regulation occurring at the transcriptional level. XDH/XO mRNA was very stable, with no indication that the rates of transcript degradation contributed to differences in mRNA accumulation or ultimate activity levels. In addition to providing information on the regulation of XDH/XO, the data presented furthers the understanding of the epithelial cell's potential to actively respond to immunomodulators associated with injury/inflammation.
Characteristics of an in vitro system of thymocyte positive selection.
Authors: Anderson G , Owen J J , Moore N C , Jenkinson E J
Published: December 2022

We investigated the phenotypic changes accompanying maturation of CD4+CD8+ thymocytes in the presence of thymic stomal cells in vitro. Many of the features that are characteristic of positive selection in vivo, such as an ordered down-regulation of CD4 or CD8 together with up-regulation of the TCR-alpha beta complex, also occurred in vitro. Transient expression of CD69 was also observed, together with activation of IL-2 and IL-4 cytokine genes, providing further evidence that positive selection in vitro involves a pattern of gene regulation comparable to that seen in vivo.
A cross-reactive idiotope on T cells from PL/J mice and Lewis rats that recognizes different myelin basic protein encephalitogenic epitopes but is restricted by TCR V beta 8.2.
Authors: Zhou S R , Whitaker J N , Han Q , Maier C , Blalock J E
Published: December 2022

Encephalitogenic T cells of both PL/J mice and Lewis rats are restricted by TCR V beta 8.2, although they recognize different epitopes in the myelin basic protein (MBP) molecule. We sought the presence of a cross-reactive idiotope (Id) in encephalitogenic T cells of Lewis rats by examining the effects of mAb F30 anti-Id, which recognized a TCR Id in PL/J T cells, on the encephalitogenic LR88L1 cell line derived from Lewis rats and specific for guinea pig MBP peptide 68-88. The LR88L1 cells were I-A restricted and TCR V beta 8.2+, and their proliferation and secretion of IL-2 and TNF-alpha induced by guinea pig MBP peptide 68-88 was inhibited by mAb F30 anti-Id. As shown by FACS analysis and by immunoprecipitation of TCR from radiolabeled LR88L1 cell lysates, the F30 anti-Id bound to the TCRs of V beta 8.2+ LR88L1 cells. In addition, TCR sequences in the F30+ population of LR88L1 cells were the same as those of encephalitogenic Lewis rat T cells published previously. The F30+ LR88L1 cells showed reduced encephalitogenicity compared with F30- or unsorted LR88L1 cells. The mechanism for this reduction by anti-Id probably resulted from the induction of anergy, in that IL-2 reversed the anti-Id effect. The control LR99L1 T cell line, also encephalitogenic, but specific for MBP peptide 87-99 and I-E, and not TCR V beta 8.2 restricted, failed to react with, or have its cytokine secretion inhibited by, mAb F30 anti-Id. These results demonstrate an interspecies cross-reactive Id expressed in common by encephalitogenic T cells that share a similar TCR, although they differ in MBP epitope specificity. These findings suggest that a common Id restricted by TCR, but less restricted by the encephalitogenic epitope, and recognized by the Id-bearing autoreactive T cells may represent an immunotherapeutic approach for treating autoimmune demyelinating diseases.
Rapamycin inhibits production of cytotoxic but not noncytotoxic antibodies and preferentially activates T helper 2 cells that mediate long-term survival of heart allografts in rats.
Authors: Ferraresso M , Tian L , Ghobrial R , Stepkowski S M , Kahan B D
Published: December 2022

Rapamycin (RAPA) induces unresponsiveness toward heart allografts by at least two mechanisms: selective production of noncytotoxic IgG2c-blocking Ab and preferential activation of Th2 cells. RAPA (0.8 mg/kg/day) delivered via a 14-day osmotic pump to Wistar Furth (WF; RT-1u) recipients prolongs Buffalo (BUF; RT-1b) heart allograft survival from a mean survival time (MST) of 6.5 +/- 0.5 days to 75.0 +/- 18.9 days (n = 18; p < 0.001), with 6 of 18 grafts beating for more than 100 days. Recipient sera or their IgG but not IgM fraction, obtained after postgrafting day 40, passively transfer the unresponsive state to sublethally irradiated secondary recipients in a dose-dependent and immunologically-specific fashion. Sera obtained after untreated WF hosts rejected BUF hearts contained IgG moieties of all subclasses that bound to class I MHC BUF epitopes. In contrast, the unresponsive sera contained predominantly non-C'-fixing IgG2c and only marginal amounts of activated (C') fixing IgG1, IgG2a, and IgG2b Ab. The transcription of IL-2, IL-4, and IL-10 mRNAs was assessed using a PCR method. There were similar increases in the levels of IL-2, IL-4, and IL-10 mRNA in heart allografts from both untreated and RAPA-treated recipients on day 5 postgrafting. In contrast, on days 60 and 300 postgrafting heart allografts from RAPA-treated unresponsive recipients showed increased levels of IL-10 and IL-4 but not of IL-2 mRNA, suggesting preferential activation of Th2 cells. Thus, RAPA treatment selectively inhibits the synthesis of C'-binding of IgG subclasses, spares the non C-binding blocking IgG2c Ab, and preferentially activates Th2 cells.
Characterization of soluble CD44 in the circulation of mice. Levels are affected by immune activity and tumor growth.
Authors: Katoh S , McCarthy J B , Kincade P W
Published: December 2022

ELISA determinations revealed substantial concentrations (0.49 to 2.10 micrograms/ml) of soluble CD44 in murine serum, with some variation among normal mouse strains. At least three species of CD44 were identified by immunoprecipitation and SDS-PAGE analysis of serum. The most prominent was indistinguishable in mobility from that extracted from normal and transformed lymphocytes and was estimated in this way to be approximately 90 kDa. A similar estimate resulted from gel filtration under nondenaturing conditions, followed by ELISA. However, lymphocyte membrane-extracted and soluble CD44 had different mobilities after treatment with neuraminidase plus O-glycosidase, and the core protein of soluble CD44 might be 17 to 20 kDa smaller than that of CD44 on lymphocyte membranes. Furthermore, an Ab to cytoplasmic residues of CD44 failed to recognize soluble CD44 recovered from the circulation or in lymphoma culture supernatants. These observations would be consistent with cleavage of CD44 from cell surfaces; and protease inhibitors slowed the loss of CD44 from cultured lymphomas. Serum CD44 levels were significantly reduced in immunodeficient CD17.SCID and BALB/c.Xid mice, and elevated in tumor-bearing mice. Mild graft-vs-host (GVH) reactions also resulted in increased concentrations of CD44, as did autoimmune disease in BXSB and MRL/lpr strains of mice. Serum with high concentrations of CD44 partially blocked the binding of one ligand, hyaluronate, to CD44-bearing hybridoma cells. The degree of inhibition was positively correlated with CD44 concentration. These findings indicate that substantial quantities of CD44 can be released into the circulation by cleavage from cell surfaces and that this process is markedly influenced by immune system activity and tumor growth. The material seemed to be intact and potentially functional.
Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.
Authors: Shultz L D , Schweitzer P A , Christianson S W , Gott B , Schweitzer I B , Tennent B , McKenna S , Mobraaten L , Rajan T V , Greiner D L
Published: December 2022

The scid mutation was backcrossed ten generations onto the NOD/Lt strain background, resulting in an immunodeficient stock (NOD/LtSz-scid/scid) with multiple defects in adaptive as well as nonadaptive immunologic function. NOD/LtSz-scid/scid mice lack functional lymphoid cells and show little or no serum Ig with age. Although NOD/(Lt-)+/+ mice develop T cell-mediated autoimmune, insulin-dependent diabetes mellitus, NOD/LtSz-scid/scid mice are both insulitis- and diabetes-free throughout life. However, because of a high incidence of thymic lymphomas, the mean lifespan of this congenic stock is only 8.5 mo under specific pathogen-free conditions. After i.v. injection of human CEM T-lymphoblastoid cells, splenic engraftment of these cells was fourfold greater in NOD/LtSz-scid/scid mice than in C.B17/Sz-scid/scid mice. Although C.B-17Sz-scid/scid mice exhibit robust NK cell activity, this activity is markedly reduced in both NOD/(Lt-)+/+ and NOD/LtSz-scid/scid mice. Presence of a functionally less mature macrophage population in NOD/LtSz-scid/scid vs C.B-17Sz-scid/scid mice is indicated by persistence in the former of the NOD/Lt strain-specific defect in LPS-stimulated IL-1 secretion by marrow-derived macrophages. Although C.B-17Sz-scid/scid and C57BL/6Sz-scid/scid mice have elevated serum hemolytic complement activity compared with their respective +/+ controls, both NOD/(LtSz-)+/+ and NOD/LtSz-scid/scid mice lack this activity. Age-dependent increases in serum Ig levels (> 1 micrograms/ml) were observed in only 2 of 30 NOD/LtSz-scid/scid mice vs 21 of 29 C.B-17/Sz-scid/scid animals. The multiple defects in innate and adaptive immunity unique to the NOD/LtSz-scid/scid mouse provide an excellent in vivo environment for reconstitution with human hematopoietic cells.