bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential uncoupling of chemoattractant receptors from G proteins in retinoic acid-differentiated HL-60 granulocytes.
Authors: Erbeck K , Klein J B , McLeish K R
Published: December 2022

The ability of HL-60 cells differentiated with DMSO or retinoic acid (RA) to serve as models for polymorphonuclear leukocytes was examined. NaF and FMLP stimulated superoxide release from both groups of cells, but the response was attenuated in RA-differentiated cells. Isolated plasma membranes from DMSO-M and RA-differentiated (RA-M) cells were used to examine receptor and guanine nucleotide binding regulatory protein (G protein) expression. Ligand binding showed that formyl peptide and leukotriene B4 receptors were present in equal density on DMSO-M and RA-M. Formyl peptide receptors on RA-M failed to demonstrate high affinity binding sites. Both G alpha i2 and G alpha i3 were present in equal density on RA-M and DMSO-M by immunoblotting. FMLP, but not leukotriene B4, failed to stimulate guanosine 5'-(gamma-thio)triphosphate binding or GTP hydrolysis and guanine nucleotides failed to inhibit FMLP binding to RA-M. Addition of solubilized membranes containing normal formyl peptide receptors to solubilized RA-M reconstituted FMLP stimulated G protein activation, whereas addition of normal G proteins had no effect. We conclude that formyl peptide, but not leukotriene B4, receptors are uncoupled from G proteins in RA-differentiated HL-60 granulocytes. Although RA-differentiated cells are an inadequate model of neutrophil transmembrane signaling, they may provide a unique model by which to study the molecular requirements for formyl peptide receptor-G protein interactions.
Thrombospondin promotes both chemotaxis and haptotaxis in neutrophil-like HL-60 cells.
Authors: Mansfield P J , Suchard S J
Published: December 2022

Differentiation of the human promyelocytic leukemia cell line HL-60 to polymorphonuclear leukocyte (PMN)-like cells with DMSO provides an important model for studying the acquisition of PMN functional responses that accompany differentiation. We showed previously that the extracellular matrix (ECM) protein thrombospondin (TSP) binds to PMN surface receptors and promotes adhesion and motility. Undifferentiated HL-60 cells did not adhere and were not motile in response to TSP, whereas cells differentiated toward PMN-like cells demonstrated both TSP-mediated adhesion and chemotaxis, with chemotaxis evident by day 2 of induction. With differentiation, a maximal response was obtained with 100 to 300 nM TSP, 10-fold lower than required for maximal PMN chemotaxis. Checkerboard analysis confirmed the directional nature of motility. mAb recognizing different domains of TSP inhibited chemotaxis, suggesting the involvement of multiple sites on TSP. Although both the NH2-terminal heparin-binding domain (HBD) and 140 kDa COOH-terminal fragment supported chemotaxis in PMN-like cells, neither fragment was as potent as intact TSP. Both pertussis and cholera toxin inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. The toxin effects did not indirectly result from elevated cAMP levels because high concentrations of either 8-bromo-cAMP or dibutyryl cAMP did not inhibit chemotaxis. TSP bound to nitrocellulose filters induced the directed migration (haptotaxis) of PMN-like cells rather than the random motility observed with PMN. Haptotaxis was stimulated by either the HBD or 140-kDa fragment and was inhibited by mAb against these two domains. Haptotaxis rather than random migration was confirmed by checkerboard analysis. Our results demonstrate that PMN-like HL-60 cells respond differently to TSP than human peripheral blood PMN. These differences may reflect 1) an aberration in HL-60 differentiation reflecting their leukemic phenotype 2) differentiation of HL-60 cells to a cell type characteristic of "activated" PMN.
Regulation of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor-1 by IFN-gamma, tumor necrosis factor-alpha, IgG aggregates, and cAMP in mouse mesangial cells.
Authors: Satriano J A , Hora K , Shan Z , Stanley E R , Mori T , Schlondorff D
Published: December 2022

The interaction of mesangial cells and monocyte-macrophages plays an important role in renal glomerular immune injury. We have, therefore, examined the regulation of two monocyte-specific cytokines, i.e., macrophage CSF-1 and monocyte chemoattractant protein (MCP-1), the product of the mouse JE gene, in mouse mesangial cells. TNF-alpha, IFN-gamma, and aggregates of IgG increased the synthesis of CSF-1 (determined by RIA) and of MCP-1 (determined by biolabeling and immunoprecipitation). Stimulation of cAMP generation by forskolin or PGE2 decreased basal CSF-1 synthesis and attenuated the responses to TNF-alpha, IFN-gamma, and IgG. Forskolin and PGE2 also decreased biolabeled MCP-1 generation after stimulation with IFN-gamma, TNF-alpha, or IgG. By Northern blot analysis steady state levels of mRNA for CSF-1 and JE/MCP-1 were increased after incubation with IFN-gamma, TNF-alpha, or IgG, and these effects were attenuated by forskolin. By using nuclear run-on assays the decrease in CSF-1 and JE/MCP-1 mRNA levels induced by stimulation of cAMP generation with forskolin was attributed to decreased transcription of these genes. Thus, agents stimulating cAMP generation, including PGE2, counterbalance the generation of CSF-1 and JE/MCP-1 in response to IFN-gamma, TNF-alpha, and IgG complexes. The locally produced CSF-1 and MCP-1 may in turn influence the interaction between mesangial cells and monocyte-macrophages in glomerular injury.
Priming to heat shock proteins in infants vaccinated against pertussis.
Authors: Del Giudice G , Gervaix A , Costantino P , Wyler C A , Tougne C , de Graeff-Meeder E R , van Embden J , van der Zee R , Nencioni L , Rappuoli R
Published: December 2022

To investigate whether and in which proportion normal individuals experience a priming to microbial heat shock proteins (hsp), the presence of antibodies to two mycobacterial hsp was tested in serum sample from 2- to 4-mo-old children before and at different times after vaccination with the trivalent vaccine against tetanus, diphtheria, and pertussis (DTP). We show that 88.9% of infants vaccinated with DTP developed antibody responses to mycobacterial hsp. Such a response was due to the whole-cell pertussis component of the vaccine, because it was not observed in infants receiving an acellular pertussis vaccine. Antibodies and cells reactive to the mycobacterial 65-kDa hsp were also found in mice immunized with DTP. Interestingly, whole-cell pertussis vaccine-induced anti-hsp antibodies cross-reacted with the Escherichia coli GroEL hsp, and at a some extent with the human 60-kDa hsp, belonging to the same hsp family. These data suggest that priming of the immune system to hsp is a common phenomenon occurring very early in life.
Anti-IL-4 diminishes in vivo priming for antigen-specific IL-4 production by T cells.
Authors: Gross A , Ben-Sasson S Z , Paul W E
Published: December 2022

Treatment of mice with neutralizing monoclonal anti-IL-4 antibodies at the time of immunization with keyhole limpet hemocyanin causes significant inhibition of priming of T cells for the production of IL-4 upon subsequent in vitro challenge. BALB/c mice received a single injection of anti-IL-4 at the time of immunization. T cells purified from spleen and lymph nodes were obtained at 6 to 7 days and at 30 to 75 days after priming. In the 6- to 7-day group, IL-4 production in response to keyhole limpet hemocyanin among the recipients of anti-IL-4 was reduced by more than twofold in four of four experiments, when low density T cells were challenged. In some, but not all, of these experiments, production of IFN-gamma was enhanced at least twofold. Measurement of frequency of IL-4-producing, keyhole limpet hemocyanin-specific T cells indicated a twofold reduction in the anti-IL-4-treated mice. Among cells obtained between 30 and 75 days after priming, production of IL-4 was diminished in four of four cases in high density cells and three of four cases in low density cells. T cells were also prepared from mice that received a secondary in vivo challenge 90 to 105 days after priming. T cells from boosted donors that had received a single injection of anti-IL-4 at the time of priming showed diminished production of IL-4 in each experiment. By contrast, treatment with anti-IL-4 at the time of secondary challenge did not diminish IL-4-producing capacity of cells from mice that were primed in the absence of anti-IL-4. These results indicate that IL-4 is important in vivo in priming T cells to develop into IL-4-producing cells and indicate an important physiologic role for IL-4 in the establishment of lymphokine-producing phenotype.
Distinction of mouse CD8+ suppressor effector T cell clones from cytotoxic T cell clones by cytokine production and CD45 isoforms.
Authors: Inoue T , Asano Y , Matsuoka S , Furutani-Seiki M , Aizawa S , Nishimura H , Shirai T , Tada T
Published: December 2022

Mouse CD8+ T cell clones could be subdivided into two subgroups by the pattern of cytokine production and CD45 isoforms that exactly corresponded with the functionally assigned cytotoxic (CTL) and suppressor (Ts) subsets. All the Ts clones invariably produced IL-10 after stimulation with immobilized anti-CD3, whereas none of CTL clones expressed mRNA of IL-10 by the same stimulation. All the CTL clones were positive for CD45RA Ag, a product of exon 4 of CD45 gene, whereas none of the Ts clones were positive for this Ag. The results are analogous to the situation within CD4+ T cell clones in which Th1 and Th2 subtypes can be distinguished by CD45 isoforms and cytokines they produce. Ts clones could suppress the proliferative responses of both Th1 and Th2 type CD4+ clones by the production of two different cytokines, IL-10 and IFN gamma, respectively, but none of the CTL clones could suppress the proliferation of CD4+ T cells. The participation of an additional nonspecific molecule in the suppression of antibody response is suggested.
Use of bispecific heteroconjugated antibodies (anti-T cell antigen receptor x anti-MHC class II) to study activation of T cells with a full length or truncated antigen receptor zeta-chain.
Authors: Wu S , Yang Y , Sadegh-Nasseri S , Ashwell J D
Published: December 2022

Ligand-induced activation of T cells involves recognition of monovalent peptide Ag complexed with a cell surface MHC-encoded molecule. In contrast, antibody-induced activation of T cells typically requires external cross-linking of the TCR. To examine the mechanisms that underlie the ability of these different stimuli to signal, we have created bispecific chimeric antibody molecules (BA) that mimic Ag in several important aspects. Anti-TCR-alpha, -beta, or anti-CD3-epsilon Fab fragments were covalently coupled to an anti-MHC class II Fab fragment. These BA elicited IL-2 production or proliferation from Ag-specific T cell hybridoma cells or splenic T cells, respectively, in the presence, but not the absence, of accessory cells expressing the appropriate MHC class II molecule. This response was prevented by soluble blocking antibodies against the TCR or MHC class II. When "presented" by MHC class II-bearing accessory cells, anti-TCR x anti-MHC class II BA, like cell surface Ag, elicited IL-2 production from T cell transfectants expressing full length TCR zeta-chain but not from otherwise identical cells expressing truncated zeta; when immobilized on a plastic surface these BA were potent stimulators that induced equal amounts of IL-2 from the same cells. Purified Ag/MHC complexes immobilized on plastic were able to induce IL-2 production from T cells expressing the full length, but not the truncated, form of zeta. We hypothesize that TCR-mediated T cell activation requires stable aggregation of the TCR. In this model, activation by mobile cell surface Ag/MHC or BA occurs in two steps, occupancy-induced TCR clustering followed by stable aggregation facilitated by the presence of a full length zeta-chain. Immobilized high affinity anti-TCR antibodies, but not low affinity Ag/MHC complexes, directly promote stable receptor aggregates, and thus would not require a full length zeta-chain.
Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains.
Authors: Cooper L J , Shikhman A R , Glass D D , Kangisser D , Cunningham M W , Greenspan N S
Published: December 2022

In this report, we examine the influence of CH domains on antibody specificity, in the context of variable epitope density on bacteria and synthetic glycoconjugates. Hybridomas secreting IgG1 and IgG2b mAb, specific for the N-acetyl-glucosamine (GlcNAc) residues of streptococcal group A carbohydrate, were previously generated from a hybridoma secreting a mouse IgG3 mAb. We show that these three mAb have identical H and L chain V domains, as determined by 1) cDNA sequencing, 2) binding to soluble Ag, and 3) binding to nine monoclonal anti-idiotopes. Nevertheless, the IgG3 mAb binds more effectively than the V region-identical IgG1 or IgG2b mAb to each of three strains of group A streptococci that display different amounts of terminal GlcNAc residues on their cell walls. The magnitude of the subclass-associated differential in binding varies with the target strain, and, whereas the IgG3 mAb binds best to the strain expressing an intermediate amount of GlcNAc, the IgG1 and IgG2b mAb and IgG3-derived F(ab')2 fragments bind best to the strain expressing the highest amount of GlcNAc. The IgG3 mAb also binds better than the IgG1 and IgG2b mAb to solid-phase GlcNAc50-BSA, but the IgG2b mAb binds best to otherwise identical conjugates with lower ratios of GlcNAc to BSA (20:1, 10:1, 5:1, and 1:1). These results suggest that epitope density can significantly influence the magnitude of IgG subclass-associated binding differences, and that structural differences in the CH regions, particularly the CH2 and CH3 domains, can influence the apparent specificities of IgG molecules for multivalent Ag.
Transfer of putative complementarity-determining region loops of T cell receptor V domains confers toxin reactivity but not peptide/MHC specificity.
Authors: Patten P A , Rock E P , Sonoda T , Fazekas de St Groth B , Jorgensen J L , Davis M M
Published: December 2022

We have used multiple-amino acid replacement mutagenesis to examine the roles of the TCR homologues of Ig complementarity-determining regions (CDR) and framework sequences in Ag-MHC and Staphylococcus aureus enterotoxin reactivity. In the three cases examined, transplantation of Ig CDR3 homologues between I-Ek-restricted TCR that recognize distinct peptides did not result in transfer of peptide reactivity. Thus the structural context of the CDR3 loops, e.g., both neighboring CDR and the V beta structure, must play a crucial, albeit supporting, role in ligand recognition. The extreme lability of this context was also shown by the fact that transplantation of the CDR1, -2, and -3 loops from the beta chain of 5C.C7 onto a V beta 1 framework failed to transfer MHC-peptide specificity even when the TCR-alpha chains were identical. In contrast, superantigen reactivity was readily transferred in several cases, with CDR2 transplants conferring strong staphylococcal enterotoxin B and A reactivity and CDR1 transplants yielding weak reactivities. This suggests that bacterial (and perhaps other) superantigens bind to many of the same regions of the TCR V beta that are believed to interact with MHC molecules. These regions of V beta may be ideal targets for superantigen binding precisely because they interact with MHC molecules and thus may be relatively conserved.
T cell-dependent IFN-gamma exerts an antiviral effect in the central nervous system but not in peripheral solid organs.
Authors: Kündig T M , Hengartner H , Zinkernagel R M
Published: December 2022

The antiviral relevance of soluble mediators that may operate in the vicinity of virus specific effector T cells was investigated. Mice were immunized with vesicular stomatitis virus (VSV) wild type (wt) and subsequently challenged with a mixture of two vaccinia recombinant viruses, one expressing the nucleoprotein of VSV (vacc-VSV-NP) the other expressing the glycoprotein of lymphocytic choriomeningitis virus (vacc-LCMV-GP). It was determined whether or not the VSV wt-induced memory T cell response that is protective against vacc-VSV-NP would inhibit growth of the nonrecognized vacc-LCMV-GP. In ovaries and testes replication of vacc-LCMV-GP was not inhibited. In contrast, the T cell response against vacc-VSV-NP nonspecifically inhibited growth of the non-recognized vacc-LCMV-GP in the central nervous system. This inhibiting effect was partly abrogated by treatment with anti-IFN-gamma antiserum but not by an anti-TNF-alpha antiserum. Similar results were obtained in VSV wt-immune H-2b mice, which eliminate vacc-VSV-NP by CD8+ T cells and in H-2k mice, which eliminate vacc-VSV-NP by a CD4+ T cell-dependent mechanism. These data suggest that a protective bystander effect mediated by soluble CD8+ and CD4+ T cell-dependent factors may be demonstrated against vaccinia virus only in an organ such as the central nervous system in which the blood-brain barrier inhibits diffusion and draining of the soluble antiviral factors released by specific effector T cells.
Type M12 protein from Streptococcus pyogenes is a receptor for IgG3.
Authors: Retnoningrum D S , Podbielski A , Cleary P P
Published: December 2022

Streptococcus pyogenes, serotype M12, was discovered to express a human IgG3 binding protein. Western blot analysis of partially purified M12 protein, exposed to IgG3 myeloma protein, showed that both M12 Ag and the receptor protein were the same apparent size. A lambda clone (lambda 4.1) containing the emm12 open reading frame expressed both the M12 Ag and the IgG3 binding protein. The emm12 open reading frame was amplified by the polymerase chain reaction and subcloned into the expression vector pJLA602. Based on Western blot analysis, one recombinant Escherichia coli (pD3) expressed M12 protein with IgG3 binding activity. This result confirmed that the M12 protein from strain CS24 is also an IgG3 receptor. Deletion analyses showed that a truncated M12 protein encoded by an internal PvuII fragment was sufficient for IgG3 binding activity. Further deletion studies suggested that the IgG3 binding domain was located in a 200 amino acid internal fragment containing two directly repeated sequences. Other experiments suggest that the receptor did not bind to the same IgG3 domain as that recognized by protein G. The M12 protein did not bind human IgG1, IgG2, IgG4, or Ig from several other animal species.
Role of leukocyte adhesion molecules in complement-induced lung injury.
Authors: Mulligan M S , Smith C W , Anderson D C , Todd R F , Miyasaka M , Tamatani T , Issekutz T B , Ward P A
Published: December 2022

The bolus i.v. infusion of cobra venom factor into rats results in acute lung injury that is neutrophil-dependent, oxygen radical-mediated, and requires CD18. In our studies a more precise definition regarding the role of beta-integrins and requirements for cytokines was obtained by the use of blocking antibodies. Lung injury was quantitated by changes in permeability (leakage of 125I-BSA) and hemorrhage (extravasation of 51Cr-RBC). In animals treated with anti-CD11a, the permeability and hemorrhage parameters were reduced by 30 and 29%, respectively. Treatment with anti-CD11b resulted in reductions in permeability and hemorrhage by 53 and 48%, respectively, whereas anti-intercellular adhesion molecule-1 reduced the parameters of injury by 60 and 75%, respectively. Not surprisingly, treatment with antibodies to very late Ag-4, TNF-alpha and IL-1 failed to show any protective effects, which contrasts to the requirements for these molecules in lung injury after deposition of IgG immune complexes. Protective interventions were associated with a reduction in lung content of myeloperoxidase. These studies indicate that, in the cobra venom factor model of acute lung injury in rats, engagement of Mac-1, lymphocyte function-associated Ag-1, and intercellular adhesion molecule-1 are essential, whereas, in contrast to other models of neutrophil mediated lung injury, cytokines (TNF-alpha and IL-1) and very late Ag-4 are not required for the full development of injury.
Accessory cell function of human eosinophils. HLA-DR-dependent, MHC-restricted antigen-presentation and IL-1 alpha expression.
Authors: Weller P F , Rand T H , Barrett T , Elovic A , Wong D T , Finberg R W
Published: December 2022

Although peripheral blood eosinophils express little of the class II MHC protein, HLA-DR, eosinophils could be induced to express HLA-DR by exposures to cytokines, including granulocyte-macrophage-CSF, IL-4, and IFN-gamma, with granulocyte-macrophage-CSF eliciting the greatest level of HLA-DR expression as assessed by flow cytometry. The capacity of HLA-DR+ eosinophils to function as APC was evaluated with blood eosinophils isolated free of mononuclear cells, cultured with granulocyte-macrophage-CSF to induce HLA-DR expression and then exposed to the Ag tetanus toxoid. HLA-DR+ eosinophils fixed with paraformaldehyde after Ag exposure stimulated T cell proliferation, whereas HLA-DR+ eosinophils fixed with paraformaldehyde before Ag exposure failed to stimulate lymphocyte proliferation. The lymphocyte proliferative responses elicited by Ag-pulsed HLA-DR+ eosinophils were inhibited by anti-HLA-DR mAb and were restricted to HLA-DR compatible lymphocytes. Moreover, eosinophils from a hypereosinophilic donor, both before and more prominently after stimulation with PMA, contained transcripts for IL-1-alpha mRNA detectable by Northern blot hybridization and in situ hybridization and expressed IL-1-alpha protein detectable by immunohistochemistry. These findings indicate that human eosinophils can process Ag, express the costimulatory cytokine IL-1-alpha, and after cytokine-elicited induction of HLA-DR expression can function as HLA-DR-dependent, MHC-restricted APC in stimulating T lymphocyte responses.
Cross-linking of Fc gamma RIIIA on natural killer cells results in tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2.
Authors: Liao F , Shin H S , Rhee S G
Published: December 2022

The Fc gamma RIIIA, which is composed of a transmembrane IgG-binding glycoprotein and either a disulfide-linked homodimer (zeta zeta, gamma gamma) or heterodimer (zeta gamma), mediates the antibody-dependent cellular cytotoxicity in NK cells. The role of phospholipase C (PLC) isozymes in Fc gamma RIIIA-mediated signal transduction was investigated. The NK cell line NK3.3 was found to contain PLC-gamma 1 and an especially high concentration of PLC-gamma 2, but PLC-beta 1 and PLC-delta 1 were not detected. Cross-linking of Fc gamma RIIIA on NK3.3 cells induced a rapid phosphorylation of PLC-gamma 1 and PLC-gamma 2 on tyrosine residues. Pretreatment of NK3.3 cells with a tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of both PLC-gamma 1 and PLC-gamma 2. These results indicate that activation of Fc gamma RIIIA on NK3.3 cells is coupled either directly or indirectly to a nonreceptor tyrosine kinase, which phosphorylates, and thereby activates PLC-gamma 1 and PLC-gamma 2.
Recombinant soluble murine IL-4 receptor can inhibit or enhance IgE responses in vivo.
Authors: Sato T A , Widmer M B , Finkelman F D , Madani H , Jacobs C A , Grabstein K H , Maliszewski C R
Published: December 2022

This study examines the effects of soluble IL-4R (sIL-4R) administration on IgE production in vivo by using an anti-IgD injection model. Anti-IgD-treated mice were given various doses of sIL-4R or anti-IL-4 mAb over a 3-day period and serum IgE levels were determined by ELISA on day 9. The sIL-4R inhibited IgE production by up to 85%. Anti-IL-4 mAb administration resulted in comparable levels of inhibition at considerably lower doses. The disparity in efficacy between sIL-4R and anti-IL-4 mAb was likely the result of differences in the biodistribution and in vivo half-life of the two IL-4-binding proteins. The specificity of the sIL-4R inhibitory effect was assessed by mixing sIL-4R with various concentrations of IL-4 before injection. Exogenous IL-4 partially overcame the inhibitory effect of high-dose sIL-4R or anti-IL-4 mAb. Unexpectedly, coadministration of suboptimal concentrations of anti-IL-4 mAb or sIL-4R with IL-4 resulted in superinduction of the IgE response. This stimulatory effect was dose dependent for both IL-4 and the IL-4 cognates and was not seen in the absence of exogenous IL-4 over the entire concentration range tested for either sIL-4R or anti-IL mAb. The results indicate that sIL-4R can block IgE secretion by neutralizing endogenous IL-4. Furthermore, sIL-4R can enhance, in a dose-dependent manner, the biologic effects of exogenously administered IL-4, presumably by altering the biodistribution of the cytokine. These findings suggest two alternative applications for cytokine-binding proteins, i.e., 1) as antagonists of biologic activities of endogenously produced cytokines and, 2) as vehicles for cytokine delivery.
Evolution of the MHC: isolation of class II beta-chain cDNA clones from the amphibian Xenopus laevis.
Authors: Sato K , Flajnik M F , Du Pasquier L , Katagiri M , Kasahara M
Published: December 2022

The amphibian Xenopus laevis is the most primitive vertebrate in which the MHC has been identified both at the biochemical and functional levels. In the present study, cDNA clones for MHC class II beta-chain genes of X. laevis were isolated using the polymerase chain reaction. In partially inbred animals with the MHC f haplotype, two distinct cDNA clones assumed to encode isotypes were identified. The deduced MHC class II beta-chains of X. laevis had structures fundamentally similar to those of their mammalian counterparts. The overall amino acid sequence identity between X. laevis and representative mammalian MHC class II beta-chains was approximately 41 to 50%. Sequence comparison of cDNA clones isolated from outbred subjects and the partially inbred frog with the MHC f haplotype showed that nucleotide substitutions are located primarily in the beta 1 domain and that non-synonymous substitutions occur more frequently than synonymous substitutions in the putative Ag-binding sites. This result indicates that, as in mammalian MHC genes, the MHC class II beta-chain genes of X. laevis are under positive Darwinian selection. Despite the fact that X. laevis is a tetraploid species, its MHC, as defined by serology, immunochemistry, and MLR, is diploidized. Family studies showed that all MHC class II beta-chain genes detectable in the genome of X. laevis segregate with serologically defined MHC haplotypes, suggesting that the second, inactivated set of MHC class II beta-chain genes was either deleted or that their sequences diverged too far to be detected by DNA hybridization. Furthermore, a nearly constant number of MHC class II beta-chain genes was detectable in most, if not all, Xenopus species with various degrees of ploidy. Thus, various polyploid species of Xenopus appear to have used a similar strategy to diploidize their MHC genes.
Integrin regulation of leukocyte inflammatory functions. CD11b/CD18 enhancement of the tumor necrosis factor-alpha responses of monocytes.
Authors: Fan S T , Edgington T S
Published: December 2022

Engagement of the integrin CD11b/CD18 (alpha M beta 2, Mac-1, CR3) on cells of monocyte (Mo) lineage has recently been demonstrated to enhance synthesis and surface expression of the integral plasmalemma receptor tissue factor. The role of cognate interactions between integrin and its ligands in the regulation of cellular responses important to inflammation can be extended to the effect on the enhancement of TNF-alpha mRNA accumulation and protein secretion by Mo once stimulated by an initial signal such as LPS. At a concentration optimal for inducing TNF-alpha responses, LPS was observed to rapidly increase by two- to threefold the surface expression on Mo of CD11b but not CD11a or CD11c. In the absence of initial signal, engagement of CD11/CD18 integrins per se failed to elicit a TNF-alpha response. In the presence of the initial transcriptional agonist LPS, both TNF-alpha mRNA expression and protein secretion were enhanced several-fold by cells adherent to a CD11b/CD18 ligand, to endothelial cells as well as engagement of CD11b/CD18 integrin by specific antibodies. This enhancement appears to be CD11b/CD18 specific and not from mere attachment or spreading. The enhancement effect after HUVEC binding was inhibited 75% by anti-CD11b mAb M1/70. These studies lead to the hypothesis that engagement of the CD11b/CD18 integrin results in the transduction of cellular signals that quantitatively enhance the expression of inflammatory mediators of Mo-mediated responses.
The serine protease cofactor factor V is synthesized by lymphocytes.
Authors: Shen N L , Fan S T , Pyati J , Graff R , LaPolla R J , Edgington T S
Published: December 2022

Ag-specific cellular immune responses result in CD4+ T cell activation, which can induce the expression of tissue factor in cells of monocyte/macrophage lineage. This results in initiation of the coagulation protease cascade, with ultimate generation of thrombin. The latter is a potent and pleiotropic mediator of cellular responses and deposition of fibrin. To explore the requirements for extravascular cellular mediation of immune effector pathways, we have searched for a cellular source of the cofactor factor Va. Factor V mRNA was identified in human lymphoid cells by using reverse transcription followed by the polymerase chain reaction (RT-PCR). We confirmed our reverse transcription-polymerase chain reaction results by an independent cloning of factor V cDNA from a T cell cDNA library. The sequence of the factor V cDNA was virtually identical to hepatic factor V mRNA sequence. A limited span of mRNA, encoding part of the connecting region of the factor V protein, was found to contain nucleotide polymorphisms based on six nucleotide substitutions. Northern blot analysis confirmed the presence of a approximately 7-kb factor V mRNA in the Hut-78* human T lymphoma cell line and, at five- to eightfold less abundance, in unstimulated lymphocytes and long term allogeneic stimulated T cells. Immunocytology with factor V mAb identified factor V intracellularly in freshly isolated T lymphocytes but not on the surface of cells. These data provide evidence for factor V transcription and biosynthesis by human lymphocytes. They provide an additional perspective on how lymphocytes may contribute to inflammatory effector functions of cellular immune responses in extravascular sites through provision of cofactors necessary for the coagulation serine protease cascade.
Human recombinant macrophage inflammatory protein-1 alpha and -beta and monocyte chemotactic and activating factor utilize common and unique receptors on human monocytes.
Authors: Wang J M , Sherry B , Fivash M J , Kelvin D J , Oppenheim J J
Published: December 2022

The human macrophage inflammatory proteins-1 alpha and -beta (MIP-1 alpha and -beta), which are also known as LD78 and ACT2, respectively, are distinct but highly related members of the chemoattractant cytokine (chemokine) family. rMIP-1 alpha and -beta labeled with 125I specifically bind to human peripheral blood monocytes, the monocytic cell line THP-1, peripheral blood T cells, and the YT cell line. Steady state binding experiments revealed approximately 3000 high affinity binding sites/cell for MIP-1 alpha on human monocytes and on THP-1 cells, with Kd values of 383 pM and 450 pM, respectively. Human MIP-1 alpha and -beta had nearly identical affinities for the binding sites and each competed equally well for binding. Human monocyte chemotactic and activating factor (MCAF), a member of the same chemokine family, consistently displaced about 25% of human MIP-1 alpha and -beta binding on monocytes but not on YT cells, which did not bind MCAF. On the other hand, human rMIP-1 alpha and -beta partially inhibited binding of radiolabeled MCAF to monocytes. Both MIP-1 alpha and -beta were chemotactic for human monocytes. Preincubation of monocytes with human rMIP-1 alpha or -beta markedly reduced cell migration towards the other cytokine, whereas preincubation with human rMCAF only partially desensitized the monocyte chemotaxis response to human rMIP-1 alpha or -beta. These data suggest the existence of three subtypes of receptors, i.e., one unique receptor shared by MIP-1 alpha and -beta, a second unique receptor for MCAF, and a third species that recognizes both MCAF and MIP-1 peptides.
Effect of transforming growth factor-beta on early and late activation events in human T cells.
Authors: Ahuja S S , Paliogianni F , Yamada H , Balow J E , Boumpas D T
Published: December 2022

Transforming growth factors-beta (TGF-beta) modulate immune responses by inhibiting the proliferation of normal T lymphocytes. To examine the mechanism(s) of this inhibition, we studied the effect of TGF-beta 1 on selected events associated with the initiation and progression of the T lymphocyte cell cycle. Human peripheral blood T cells were stimulated with anti-CD3 mAb, PHA, PMA, or ionomycin, alone or in combination. TGF-beta 1 (0.5 to 10 ng/ml) partially inhibited the tyrosine phosphorylation of a 100-kDa protein, but not the calcium influx when cells were stimulated via TCR. Nuclear transcription of early activation genes (c-fos, c-jun, and c-myc) as determined by nuclear run-off assays, and steady state mRNA levels and/or protein products of intermediate activation genes (IL-2, IL-2R alpha, IL-2R beta, and transferrin receptor) were not affected by TGF-beta 1. Total cellular RNA synthesis and cell size after T cell stimulation were also not affected by TGF-beta 1. However, TGF-beta 1 inhibited the IL-2-dependent proliferation of Con A lymphoblasts by -50%. This inhibition was associated with the down-regulation of IL-2-mediated tyrosine phosphorylation of proteins of 120, 100, 85, 75, and 50 kDa. TGF-beta 1 also inhibited the IL-2-dependent phosphorylation of the retinoblastoma susceptibility gene product, which plays an important role in cell cycle progression. These results suggest that TGF-beta 1 inhibits T cell proliferation by down-regulating predominantly IL-2-mediated proliferative signals.
A novel T cell activation antigen identified by monoclonal IMN3.1 antibody and expressed preferentially on human T cells susceptible to apoptotic cell death.
Authors: Uehara T , Miyawaki T , Natsuume-Sakai S , Nibu R , Hasui M , Yachie A , Shimizu S , Taniguchi N
Published: December 2022

When cultured without appropriate growth factors, most of activated (CD45RO+) T cells expanded in acute EBV-induced infectious mononucleosis (IM) easily die via an apoptotic cell death mechanism, indicating one Ag-driven selection in the periphery. In this work, we attempted to raise the mAb against cell surface molecules, preferentially expressed on T cells entering apoptosis, by immunizing PBMC from an acute IM patient. We obtained one mAb, termed IMN3.1, that could define clearly the expansion of activated (CD45RO+) T cells in the blood of acute IM patients. In contrast to its intense expression on IM T cells, the IMN3.1-reactive Ag was only dimly expressed on CD45RO+ (memory or previously activated) populations of T cells from normal individuals. Although naive (CD45RO-) populations of T cells usually lacked IMN3.1 Ag, this Ag was inducible relatively late after in vitro activation of naive T cells. The cellular distribution and molecular characterization of IMN3.1-reactive Ag suggested that IMN3.1 mAb appeared to recognize a novel activation-associated cell surface determinant of about 120 kDa m.w., which might be predominantly expressed on apoptosis-prone T cell lineage cells, such as IM T cells, thymocytes, cytokine-dependent T cell lines, and anti-Fas-sensitive T cell lines.
A protein of the AP-1 family is a component of nuclear factor of activated T cells.
Authors: Castigli E , Chatila T A , Geha R S
Published: December 2022

Nuclear factor of activated T cells (NF-AT) is a transcriptional activator involved in the induction of IL-2 gene expression. The response element for NF-AT is a sequence localized between -285/-254 in the IL-2 regulatory region. The composition of NF-AT protein is still not fully elucidated. We demonstrate that, in normal human T cells, an AP-1 protein is a component of the NF-AT protein complex. This was evidenced by the ability of the AP-1 site to compete with the NF-AT site for binding to NF-AT and by the capacity of immobilized anti-Jun and anti-Fos antibodies to deplete NF-AT-binding activity from nuclear extracts of activated T cells. There was no detectable binding of in vitro translated Jun/Fos heterodimer (AP-1) to the NF-AT sequence, and the NF-AT sequence was unable to inhibit the binding of Jun/Fos to the AP-1 sequence. The presence of an AP-1 protein in the NF-AT protein complex may regulate NF-AT-binding activity through protein-protein interaction.
Endogenous Ig production in mu transgenic mice. I. Allelic exclusion at the level of expression.
Authors: Imanishi-Kari T , Bandyopadhay S , Busto P , Huang C A
Published: December 2022

Our findings indicate that allelic exclusion is maintained in B cells that successfully rearrange and express and endogenous H chain gene, despite carrying a functionally rearranged H chain transgene (17.2.25 mu a). Cloned hybridomas having a functionally rearranged endogenous H chain gene as well as the transgene produce only endogenous gene products. Some of these hybridoma cultures, upon continuous growth, will secrete transgene as well as endogenous gene products. However, the two H chain polypeptides appear to be made by different cells. In each of three cases of such "double producer" lines examined, further subcloning at this time reveals two types of clones: those that secrete only transgene and those that secrete only endogenous H chains. The clones producing transgene product have lost the functionally rearranged endogenous H chain genes, whereas the clones producing endogenous gene products still contain both transgene and endogenous functionally rearranged H chain genes. These results indicate that hybridomas expressing endogenous Ig have transgene copies that are potentially functional but are reversibly silenced by an unknown mechanism and suggest that inhibition of transgene expression may be mediated by endogenous Ig gene expression.
Different appearance of gamma delta T cells during salmonellosis between Ityr and Itys mice.
Authors: Emoto M , Naito T , Nakamura R , Yoshikai Y
Published: December 2022

Ity gene, which is believed to be a single gene on proximal mouse chromosome 1, determines susceptibility to Salmonella infection. We have recently demonstrated that the gamma delta T cells specific for purified protein derivative appear at the early stage after infection with Salmonella choleraesuis strain 31N-1 (avirulent strain) in mice. In the present study, we compared the appearance of the gamma delta T cells in Ityr and Itys mice after infection with S. choleraesuis in an attempt to elucidate the role of the gamma delta T cells in the in vivo effect of the Ity locus on murine salmonellosis. The numbers of gamma delta T cells in the peritoneal cavity and liver were significantly larger in DBA/2 (Ityr) mice than those in BALB/c (Itys) mice after infection with a sublethal dose of S. choleraesuis. C.D2 Ityr mice, which are BALB/c mice with Ityr allele, also showed a significant increase in the number of the gamma delta T cells at the early stage after infection. Similar results in different appearances of the gamma delta T cells could be obtained between BALB/c and DBA/2 mice after infection with Mycobacterium bovis, bacillus Calmette-Guérin. The peritoneal adherent cells of DBA/2 mice infected with S. choleraesuis, which expressed a large amount of heat shock/stress protein (hsp) homologous to the mycobacterial 65-kDa hsp, induced significantly higher level of expansion of the peritoneal gamma delta T cells in normal mice in vivo and in vitro than did those of BALB/c mice infected with S. choleraesuis, which expressed a lesser amount of the hsp. These results suggest that the Ity locus may be closely related to the expression of hsp by macrophages after infection with Salmonella, which in turn stimulate the gamma delta T cells in the host during an early phase of salmonellosis.
Macrophage-dependent candidacidal mechanisms in the murine system. Comparison of murine Kupffer cell and peritoneal macrophage candidacidal mechanisms.
Authors: Redmond H P , Shou J , Gallagher H J , Kelly C J , Daly J M
Published: December 2022

Candida albicans infection is common in immunocompromised patients. The role of fixed tissue macrophages (M phi), including Kupffer cells (KC) and peritoneal macrophages (PM phi), in host defense against C. albicans is unclear. This study examined murine M phi candidacidal mechanisms and evaluated the in vitro role of the macrophage-activating factor IFN-gamma in augmenting these mechanisms. The effect of in vivo administration of IFN-gamma on survival after lethal C. albicans challenge in the murine system was also assessed. Percent PM phi and KC ingestion of C. albicans were similar. Prior opsonization of Candida increased the percentage of M phi ingestion of this pathogen. PM phi and KC phagocytic function was similar for both nonopsonized and opsonized C. albicans, but KC demonstrated markedly decreased ability to kill this pathogen (O2-, Candida killing). IFN-gamma enhanced KC and PM phi candidacidal activity. PM phi and KC Ag presentation was increased in early Candida infection, but diminished in established infection, when the majority of animals died. C. albicans failed to elicit significant amounts of either IL-1 or TNF compared with LPS stimulation of PM phi and KC in vitro. IFN-gamma treatment in vivo was associated with significantly improved survival (p < 0.01).
Comparative analysis of the human macrophage inflammatory protein family of cytokines (chemokines) on proliferation of human myeloid progenitor cells. Interacting effects involving suppression, synergistic suppression, and blocking of suppression.
Authors: Broxmeyer H E , Sherry B , Cooper S , Lu L , Maze R , Beckmann M P , Cerami A , Ralph P
Published: December 2022

Macrophage inflammatory protein (MIP)-1 alpha, part of a family termed chemokines, has been implicated in suppression of hemopoietic stem and progenitor cell proliferation. The chemokine family has been organized into two subgroups with MIP-1 alpha, MIP-1 beta, macrophage chemotactic and activating factor (MCAF) and RANTES belonging to one subgroup, and GRO-alpha, MIP-2 alpha (GRO-beta), MIP-2 beta (GRO-gamma), platelet factor 4 (PF4), IL-8, and neutrophil activating peptide (NAP)-2 belonging to the other. These molecules were evaluated for effects on colony formation by human bone marrow multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. None of the chemokines stimulated colony formation in the absence of CSF, or influenced colony formation stimulated by a single growth factor such as granulocyte-macrophage-CSF or erythropoietin. However, MIP-1 alpha, MIP-2 alpha, PF4, IL-8, and MCAF suppressed in dose-response fashion colony formation of immature subsets of myeloid progenitor cells stimulated by GM-CSF plus steel factor. Effects were apparent on low density and CD34 HLA-DR(+)-sorted marrow cells in which up to 88.4% of the cells were composed of progenitor cells, suggesting direct effects on the progenitors themselves. Up to 2500-fold less of each chemokine could be used to demonstrate synergistic suppression when any two of these five chemokines were used together at low concentrations, effects also apparently directly on the progenitors. In contrast, MIP-1 beta, MIP-2 beta, GRO-alpha, NAP-2, and RANTES were not suppressive nor did they synergize with MIP-1 alpha, MIP-2 alpha, PF4, IL-8, or MCAF to suppress. However, a fivefold excess of MIP-1 beta blocked the suppressive effects of MIP-1 alpha. Similarly, a fivefold excess of either MIP-2 beta or GRO-alpha blocked the suppressive effects of IL-8 and PF4. These suppressing, synergizing and blocking effects may be of relevance to blood cell regulation.
Integrin alpha E beta 7 mediates adhesion of T lymphocytes to epithelial cells.
Authors: Cepek K L , Parker C M , Madara J L , Brenner M B
Published: December 2022

Intestinal intraepithelial lymphocytes (iIEL) are a distinct subpopulation of T lymphocytes diffusely distributed in the epithelium. Because > 95% of these iIEL express the integrin alpha E beta 7, we reasoned that this integrin might mediate the localization of iIEL to the epithelium. We report that cultured iIEL bound to mucosal epithelial cells derived from either breast or intestine. This binding was dependent on the presence of divalent cations, and was blocked by mAb specific for two integrins, alpha E beta 7 and alpha L beta 2. After TGF-beta 1 treatment, cultured iIEL cell lines expressed alpha E beta 7 at levels similar to those found on iIEL in vivo. Under these conditions this integrin appeared to predominate in mediating the adhesion of iIEL to epithelial cells. Although alpha L beta 2 blocked the adhesion of iIEL to both endothelial and epithelial cells, alpha E beta 7 blocked binding only to epithelial cells suggesting that the ligand for alpha E beta 7 is not expressed on IL-1 beta-treated cultured endothelial cells. iIEL did not readily bind to the apical surface of confluent polarized epithelial cell monolayers. However, iIEL did bind to these cells when tight junctions were disrupted, a treatment that allows redistribution of proteins compartmentalized to the basolateral surface. This binding was also blocked by mAb to alpha E beta 7. In addition, in vitro coculture of iIEL with polarized epithelial cells resulted in basolateral localization of the iIEL. Thus, it is likely that expression of the alpha E beta 7 ligand is restricted to the basolateral membrane of polarized epithelial cells. Because alpha E beta 7 appears to be a predominant integrin on freshly isolated iIEL, we hypothesized that alpha E beta 7-mediated adhesion may be particularly important in cell-to-cell interactions between T cells and epithelial cells in vivo.
Cytokines and dexamethasone modulation of IL-1 receptors on human neutrophils in vitro.
Authors: Shieh J H , Peterson R H , Moore M A
Published: December 2022

Modulation of IL-1R on human neutrophils was investigated after in vitro treatment of cells with human recombinant (hr) granulocyte (G)-CSF, hrgranulocyte-macrophage (GM)-CSF, hrCSF-1, hrIL-1 alpha, hrIL-2, hrIL-3, hrIL-4, hrIL-5, hrIL-6, hrIL-7, transforming growth factor-beta 1, or hrTNF-alpha. At 4 degrees C, 125I-IL-1 binding was competed by IL-1 but not by other cytokines tested. At 37 degrees C, GM-CSF, TNF-alpha, and IL-1 decreased 125I-IL-1 binding in a dose-dependent manner. Kinetic studies showed that GM-CSF reduced > 45% IL-1 binding within 15 min but later (8 h) produced a > 2-fold increase. In contrast, TNF decreased > 85% IL-1 binding within 15 min with a recovery of > 80% relative to that of control after 24 h. Scatchard analysis revealed that TNF or GM-CSF down-modulation of IL-1 binding was due to a decrease of IL-1R number. Further studies showed that dexamethasone and GM-CSF (or G-CSF) synergistically increased IL-1 binding after 8 h. This synergistic modulation was a cytokine dose- and time-dependent process, and was due to an increase in IL-1R numbers rather than a change in binding affinity. In addition, human bone marrow neutrophils, cord blood neutrophils, and several human hematopoietic cell lines (HL-60, U-937, and AML-193) responded to dexamethasone and GM-CSF (or G-CSF) with a superadditive increase in IL-1 binding. Because mammalian systems respond to bacterial endotoxins with secretion of TNF, IL-1, glucocorticoids, G-CSF and GM-CSF, our results shed additional light on this highly regulated cytokine network and revealed a novel role for GM-CSF.