bluuebunny/crossref_metadata_2025 huggingface dataset:
Switch circles from IL-4-directed epsilon class switching from human B lymphocytes. Evidence for direct, sequential, and multiple step sequential switch from mu to epsilon Ig heavy chain gene.
Authors: Zhang K , Mills F C , Saxon A
Published: December 2022

Ig isotype switch via deletional recombination is accompanied by excision of the intervening DNA between the two switch regions. The excised DNA is looped out as extrachromosomal circular DNA or switch circle. Such switch circles have been isolated and characterized in mice. We investigated deletional recombination in human B cells undergoing Ig isotype switching to demonstrate whether switch circles are also excised, and to thereby gain insight into the processes involved in human isotype switching. We characterized the deleted switch circular DNA from IL-4 directed mu to epsilon switching in polyclonal human B lymphocytes. By using two sets of specially designed PCR primers, we amplified switch circle fragments representing switch circles resulting from mu to epsilon direct switching and mu-gamma-epsilon sequential switching. The PCR-amplified products were subcloned by a TA cloning strategy and resulting clones were screened by hybridization with a 5'S epsilon probe. Sequence analysis of the positive clones revealed that all clones representing mu to epsilon direct switching indeed had 5'S epsilon directly joined to 3' S mu. Most clones representing mu-gamma-epsilon sequential switching showed 5'S epsilon joined to 3' S gamma as expected. However, two clones contained S mu and S alpha 1 sequences interposed between 5'S epsilon and 3'S gamma, respectively. These data demonstrate that switch circles are excised during human B cell isotype switching, and that IL-4 directed epsilon class switching is accomplished by 1) direct mu to epsilon switching, 2) sequential mu-gamma-epsilon switching, and 3) double sequential mu-alpha-gamma-epsilon switching.
Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.
Authors: Ernst C A , Zhang Y J , Hancock P R , Rutledge B J , Corless C L , Rollins B J
Published: December 2022

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine-beta (or C-C) family of cytokines. Murine MCP-1, first identified as the JE gene, differs from human MCP-1 in molecular size and extent of glycosylation. We have used Chinese hamster ovary cells to express recombinant murine MCP-1 and find that its predominant form is a microheterogeneous protein of M(r) approximately 25,000. Most of MCP-1's microheterogeneity is due to variable amounts of sialic acid that are terminally attached to a constant number of O-linked oligosaccharide chains per molecule. This carbohydrate, along with a small amount of N-linked carbohydrate, accounts for 50% of the apparent molecular size of murine MCP-1 and is not required for in vitro monocyte chemoattractant activity. Mutational analysis shows that most of the carbohydrate is added to a 49-amino acid C-terminal domain that is not present in human MCP-1 and is not required for in vitro biologic activity, suggesting that murine MCP-1 consists of an N-terminal domain containing monocyte chemoattractant activity and a heavily glycosylated C-terminal domain of as yet unknown function. MCP-1 produced in COS cells contains a small amount of sulfate, but Chinese hamster ovary-produced MCP-1 does not. The absence of sulfate does not alter MCP-1's in vitro chemoattractant properties. In vitro, highly purified murine MCP-1 attracts monocytes, but not neutrophils, with a specific activity similar to human MCP-1 (EC50 approximately 0.5 nM). Equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM. In vivo, injection of up to 1 micrograms murine MCP-1 in a variety of murine strains induces the appearance of a sparse mixed inflammatory infiltrate. The disparity between MCP-1's in vitro and in vivo effects suggests that other factors may be required to elicit a full-blown monocyte chemotactic response to MCP-1 in vivo.
TNF induces internalization of the p60 receptor and shedding of the p80 receptor.
Authors: Higuchi M , Aggarwal B B
Published: December 2022

As is true for other peptide hormones, TNF causes the down-modulation of its own receptor. The process by which down-regulation occurs and the particular role of each of two recently identified receptors, however, are not understood. In this report we used Abs specific to p60 and p80 TNFR types to examine the ligand-induced down-regulation in histiocytic cell line U-937. These cells express both types of TNFRs, but the amount of p80 is two to three times greater than that of the p60 receptor. Treatment of U-937 cells with the ligand led to maximum down-modulation of TNFR within 30 min, and this decrease was found to be a result of receptor number and not affinity. When examined for the receptor type, approximately 90% of the p60 receptor and 35% of the p80 receptor was down-regulated by the cytokine. Rapid internalization of TNF in U-937 cells is mediated through p60, because this process was inhibited only by anti-p60 Ab and was also inhibited in cells (PMA-pretreated) that primarily express the p80 receptor. In contrast to p60, we observed that the ligand-induced shedding of the p80 receptor into the medium results in the down-regulation. Interestingly, however, the signal for shedding of the p80 receptor appears to be mediated through the p60 receptor, because anti-p60 Ab inhibited the shedding. Overall, our results provide evidence that ligand-induced down-modulation of TNFR is a result of the internalization of p60 and of the shedding of the p80 receptor and that the signaling for both is mediated through the p60 receptor.
In vivo or in vitro anti-CD3 epsilon chain monoclonal antibody therapy for the prevention of lethal murine graft-versus-host disease across the major histocompatibility barrier in mice.
Authors: Blazar B R , Taylor P A , Vallera D A
Published: December 2022

We explored the use of a single in vivo dose of an anti-CD3 epsilon mAb for graft-vs-host disease (GVHD) prevention in mice. Anti-CD3 epsilon (145-2C11) given on the day of bone marrow (BM) transplant was highly effective in preventing GVHD in a dose-dependent manner. However, mice experienced acute toxicity as measured by early post-BM transplantation mortality, severe weight loss, and cachexia, which is likely due to T cell-dependent lymphokine production. Donor T cells were partly responsible because toxicity still occurred in mice injected with an allelic specific anti-CD3 epsilon mAb that only reacted with the donor strain. Toxicity was also dependent on the number of radioresistant host T cells present at the time of transplant, because toxicity still occurred when recipients were depleted of mature T cells in vivo with anti-CD4+ anti-CD8 mAbs, and were given T cell-depleted BM from SCID mice. Toxicity was eliminated by treating BM with anti-CD3 epsilon mAb in vitro before BM transplant instead of administering the mAb in vivo. However, GVHD protection was not complete. Direct comparison of the in vivo and in vitro approaches revealed that the in vivo administration of anti-CD3 epsilon mAb was more effective for GVHD protection and that a combination of the approaches was not beneficial. Flow cytometry studies were performed to assess the degree of CD3 epsilon chain modulation and T cell depletion obtained with in vitro vs in vivo anti-CD3 epsilon chain mAb. In mice given syngeneic BM transplants to avoid the confounding effects of GVHD, we found that in vivo anti-CD3 epsilon mAb administration induced a more prolonged period of CD3 epsilon chain modulation and/or T cell depletion than either the in vitro approach or a third approach that involved anti-CD3 epsilon F(ab')2 fragments administered in vivo. These data indicate that targeting the Ag-specific TCR, especially in such a way as to avoid toxicity associated with T cell activation, may provide an important key for developing future GVHD therapies.
Aged T cells are hyporesponsive to costimulation mediated by CD28.
Authors: Engwerda C R , Handwerger B S , Fox B S
Published: December 2022

The ability of T cells to proliferate in response to a number of stimuli is impaired in healthy, aged individuals. T cells from young (2 to 4 mo) and aged (20 to 26 mo) mice were stimulated with immobilized anti-CD3 epsilon-chain mAb and soluble anti-CD28 mAb. T cells from young and aged mice proliferated comparably in response to anti-CD3 epsilon mAb alone. However, aged T cells were significantly less responsive to costimulation by anti-CD28 mAb. This decreased response of aged T cells was seen at all dosages tested of anti-CD3 epsilon and anti-CD28 mAbs and in the presence and absence of APC. Similar results were observed when costimulation was provided by B7-transfected L cell fibroblasts. T cells from young and aged mice had comparable expression of CD28 by flow cytometry, both before and after stimulation. The defect in response to CD28 was seen both in CD4+ and CD8+ T cells and in CD44lo (naive) and CD44hi (memory) T cells. The impaired response to costimulation mediated by CD28 on T cells from aged mice may be an important factor in reduced T cell responses associated with aging.
Enkephalin-containing peptides processed from proenkephalin significantly enhance the antibody-forming cell responses to antigens.
Authors: Hiddinga H J , Isaak D D , Lewis R V
Published: December 2022

Highly conserved enkephalin containing peptides (ECPs) are selectively processed from proenkephalin, which is synthesized in both the neuroendocrine and immune systems. The reported regulatory effects within the central nervous system and the biologic release patterns from both activated lymphocytes and stimulated adrenal chromaffin cells suggest the ECPs may act as regulatory factors of the immune system. We tested the effects of three of the ECPs, Peptides F, E, and B, on the in vitro Ab-forming cell (AFC) response murine splenocytes to antigenic challenge. In contrast to the immunosuppressive effects of the pentapeptide enkephalins, physiologic concentrations of the ECPs significantly enhanced the AFC response to both T cell-dependent and T cell-independent Ags. The effects are not sensitive to competition by the opiate receptor antagonist, naloxone, suggesting cell surface interactions that do not involve classical opiate receptors. These studies provide evidence that the effects are mediated through T cells rather than B cells. Peptide F-treated splenocytes also showed a significant enhancement of the AFC response to suboptimal Ag concentrations, suggesting a mechanism of action in which the ECPs may act to lower the threshold of activation of the effector cell. These results suggest that the ECPs are physiologically important modifiers of the humoral immune response. Given their release patterns and demonstrated action on the in vitro immune response, the proenkephalin-derived ECPs have the potential to be involved in both paracrine and autocrine regulatory networks within the immune system and as a positive immunoregulatory effect from the neuroendocrine system.
Induction of B cell and T cell tolerance in vivo by anti-CD23 mAb.
Authors: Morris S C , Lees A , Holmes J M , Jeffries R D , Finkelman F D
Published: December 2022

T cell tolerance can be induced by B cell presentation of Ags to naive T cells. To further characterize this mechanism of T cell tolerance induction, we have investigated the effects of injecting mice with an intact rat IgG2a Ab, which binds to the B cell low-affinity Fc epsilon receptor (CD23), on the responsiveness of B cells and T cells to rat IgG2a. Our observations indicate that 1) intravenous, subcutaneous, or intraperitoneal injection of this Ab induces antigen-specific B cell and T cell tolerance; 2) both forms of tolerance are induced more completely by injection of rat IgG2a anti-CD23 mAb than by injection of an equal dose of a control rat IgG2a Ig; and 3) reduced responsiveness to Ag is seen as early as 1 to 3 days after anti-CD23 mAb injection and reaches maximum levels by 7 days after injection. Although tolerance induced by the injection of soluble proteins has been reported to be characterized by reduced production of IL-2 and IFN-gamma, but normal production of IL-4, injection of mice with rat IgG2a anti-mouse CD23 mAb greatly decreases the IL-4 response to a rat IgG2a immunogen that normally induces a large IL-4 response.
Human primary immune response in SCID mice engrafted with human peripheral blood lymphocytes.
Authors: Sandhu J , Shpitz B , Gallinger S , Hozumi N
Published: December 2022

Severe combined immune deficient (SCID) mice were engrafted with human peripheral blood lymphocytes (Hu-PBLs) after treatment with anti-asialo GM-1 antiserum and radiation. This pretreatment facilitates high level of Hu-PBL engraftment in the SCID mice spleen. The next day, the Hu-PBL-SCID mice were immunized with either keyhole limpet hemocyanin (KLH), or carbohydrate Ag STn (AcNeu-alpha 2-alpha 6-Gal NAc-0) conjugated to KLH or protein of circumsporozoite malaria parasite (CSP), in a mixture of complete and incomplete Freund's adjuvant (1:10 v/v). The mice were bled 14 to 16 days after immunization, and the sera analyzed for STn-, KLH-, and CSP-specific human IgG and IgM Abs. The results showed that the immunized animals had a significantly higher titer of Ag-specific human IgG and IgM Abs compared to the control Hu-PBL-SCID mice. No significant Ab cross-reactions were detected between sera from KLH-, STn-, and CSP-vaccinated Hu-PBL-SCID mice. Depletion of either human CD4+ or CD8+ cells, in Hu-PBL-SCID mice, showed that CD4+ cells were essential for the primary immune response, but depletion of CD8+ cells had no influence on the titer of Ag-specific human IgG and IgM Abs.
Human CD45RC specificity. A novel marker for T cells at different maturation and activation stages.
Authors: Zapata J M , Pulido R , Acevedo A , Sánchez-Madrid F , de Landázuri M O
Published: December 2022

The different isoforms of CD45 seem to play an important role in thymocyte maturation and T cell activation and function. To investigate the particular contribution of CD45-exon C (exon 6) to human T cell development, we first investigated the expression of the CD45-exon C isoforms on different human lymphoid cell populations. A wide panel of mAbs against the different isoforms of CD45 was generated by immunizing mice with purified human CD45. Two of these mAbs (RP1/12 and RP2/19) selectively recognized mouse 300-19 pre-B cells that had been transfected with CD45 cDNA that contained exon C, suggesting that epitopes recognized by these mAbs are dependent on the expression of exon C. Immunofluorescence analysis of PBLs showed that CD45RC is expressed at high levels on B, NK, and CD8+ T cells, although a subset of CD4+ T cells was found to be negative. Activation of PBMCs and purified NK cells resulted in down-regulation of CD45RC, which took place in a coordinate manner involving both down-regulation of the expression of CD45RA and CD45RB and up-regulation of CD45R0. Two-color immunofluorescence analyses showed that thymocytes expressing CD45RC were CD1-to lo, CD3hi and HLA class Ihi, typical markers of mature thymocytes. Accordingly, immunohistochemical analysis of a normal thymus showed that whereas the anti-CD45RA mAb stained only a subset of medullary thymocytes, the anti-CD45RC mAb stained the majority of medullary thymocytes. Thymocytes that bear CD45RA also express CD45RC, but do not express CD45RO; a subset of CD45 RA- RO+ thymocytes was also detected. Immunoprecipitation of CD45 molecules from both PBMC and thymocyte cell lysates with mAbs against the different specificities of this molecule revealed that the RC determinants are found on CD45 polypeptides of 220 and 205 kDa that also bear RB or both RB and RA determinants, respectively. Together these results support both the existence of a tight regulation of the expression of CD45 specificities during lymphocyte activation and thymocyte maturation and also that CD45RC is selectively expressed by mature medullary thymocytes during T cell ontogeny.
Immunogenic and antigenic properties of an HIV-1 gp120-derived multiple chain peptide.
Authors: Kelker H C , Schlesinger D , Valentine F T
Published: December 2022

An HIV-1 envelope protein gp120-derived monomeric peptide (amino acid residues 419-439) and its homologous multiple chain peptide (MCP) construct were compared for immunogenicity in mice. The Abs stimulated by the MCP recognized epitopes on the MCP that were not present on the homologous monomer. The anti-419-439 MCP sera recognized a conformational determinant on the native envelope glycoprotein, as indicated by: 1) detection of native but not denatured recombinant envelope glycoprotein by ELISA and dot blot and 2) reaction with infected cell lines expressing gp120 on their surface as detected by flow cytometry. In contrast, the anti-monomer sera were highly specific for the monomer and recognized the envelope glycoprotein at lower titers. The low reactivity of the anti-monomer sera with the envelope glycoprotein was not decreased by denaturation. Reciprocally, murine antiserum to HIV-1 envelope glycoprotein gp160 recognized the MCP construct but not the homologous monomeric peptide. The data indicate that the MCP construct forms additional antigenic determinants not present on the homologous monomer, and that the anti-419-439 MCP Abs recognize a conformational determinant on the envelope glycoprotein not recognized by Abs against the homologous monomer. Furthermore, antisera against another envelope-derived MCP (amino acid residues 105-117) also recognize conformational determinants on the envelope glycoprotein, whereas antisera against the homologous monomeric peptide do not.
BUB/BnJ (H-2q) is a TCR deletion mutant mouse strain (TCR V beta a, KJ16-) that is susceptible to type II collagen-induced arthritis.
Authors: Ortman R A , Holderbaum D , Qu X M , Banerjee S , Haqqi T M
Published: December 2022

Type II collagen-induced arthritis (CIA) in mice is an animal model of autoimmune inflammatory arthritis. Arthritis is induced in susceptible strains of mice (H-2q and H-2r) after immunization with heterologous or autologous type II collagen in CFA. Induction of CIA is T cell dependent and a restricted usage of TCR V beta genes has been found in the arthritic joints and lymph nodes of mice with CIA. However, genes within the MHC are not the only determinants of susceptibility to CIA as SWR/j, AU/ssJ (both H-2q) and RIIIS/J(H-2r) mice are resistant to the induction of CIA. These strains of mice are TCR V beta chain genes deletion mutants (TCR V beta a and TCR V beta c haplotypes) and it was hypothesized that these mice are resistant to CIA because of the absence of a particular set of V beta TCR genes that are genomically deleted in these strains of mice. We now show that BUB/BnJ mice (H-2q) are T cell subsets deficient because of the genomic deletion of TCR V beta 5, 8, 9, 11, 12, and 13 sub-families. Our data demonstrate that despite the deficiency in T cell subsets from genomic deletion of TCR V beta genes, BUB mice are highly susceptible to the development of CIA. These results indicate that genomic deletion of certain TCR V beta genes alone is not in itself sufficient to confer resistance to CIA. These results further suggest that other unknown gene(s) must also contribute to the induction of CIA.
Phenotypic and functional cellular differences between human CD3- decidual and peripheral blood leukocytes.
Authors: Deniz G , Christmas S E , Brew R , Johnson P M
Published: December 2022

CD3- leukocyte clones derived from human decidualized endometrial tissue of first trimester pregnancy have been compared with CD3- PBL clones. Most CD3- decidual granulated leukocyte (DGL) clones were CD16- CD56+, whereas most CD3- PBL clones were CD16+ CD56+. CD3- DGL and PBL clones, whether CD16+ or not, showed MHC-nonrestricted NK cell activity. However, CD3- CD16- DGL clones had low cytotoxic activity against the NK-resistant cell line BSM, whereas CD3- CD16+ DGL and CD3- PBL clones were strongly cytotoxic. Cytolytic activity has also been investigated in respect of target cell HLA-G expression, because this nonpolymorphic class I MHC molecule is expressed selectively by invasive fetal cytotrophoblast. Class I HLA Ag loss cell mutants were killed efficiently by CD3- DGL clones. Expression of transfected HLA-B8 increased their sensitivity to lysis by most CD3- DGL clones, whereas expression of transfected HLA-G commonly led to decreased target cell killing. In addition, the effects of uncloned CD3- DGL on the one-way MLR have been examined. These cells were very poor responders and, unless cultured to induce expression of class II MHC molecules, were also very poor stimulators. When fresh CD3- DGLs were added as third-party cells, either autologous or allogeneic to responder cells, [3H]TdR incorporation was decreased in the MLR. Thus, CD3- DGL clones express MHC-nonrestricted cytolytic activity, notably against HLA-negative cells, but expression of HLA-G offers protection to target cells. In addition, CD3- DGL may function to suppress allogeneic responses.
Activation of a novel serine/threonine kinase that phosphorylates c-Fos upon stimulation of T and B lymphocytes via antigen and cytokine receptors.
Authors: Nel A E , Taylor L K , Kumar G P , Gupta S , Wang S C , Williams K , Liao O , Swanson K , Landreth G E
Published: December 2022

Ligation of Ag receptors in T and B lymphocytes initiates signal transduction cascades which alter the expression of genes that regulate cellular proliferation and differentiation. The transmission of signals from the membrane to the nucleus is mediated principally through the action of protein tyrosine and serine/threonine kinases. We have identified and characterized a novel serine/threonine kinase that phosphorylated the proto-oncogene product, c-Fos, and is termed Fos kinase. Fos kinase was rapidly activated after ligation of the CD3 and CD2 receptors in Jurkat and normal human T lymphocytes and in response to IL-6 and anti-IgM in the human B cell lines AF10 and Ramos, respectively. The phorbol ester, PMA, was also a potent inducer of Fos kinase activity in all of the above populations, suggesting that PKC plays a role in the regulation of this enzyme. Fos kinase phosphorylates c-Fos at a site near the C-terminus, as well as a peptide derived from this region (residues 359-370, RKGSSSNEPSSD), and Fos peptide competitively inhibited c-Fos phosphorylation. Fos kinase was shown to be distinct from other identified serine/threonine kinases, including protein kinase A, protein kinase C, casein kinase II, MAP kinases, p70S6K and p90RSK. Fos kinase was purified by anion exchange chromatography and exhibited an apparent M(r) = 65,000 and isoelectric point = 6.1. Fos kinase may play a role in transcriptional regulation through its capacity to phosphorylate c-Fos at a site required for expression of the transcriptional transrepressive activity of this molecule. Moreover, its rapid activation suggests it may have a wider role within signal transduction cascades in lymphocytes.
Efficient induction of immunoglobulin production in neonatal naive B cells by memory CD4+ T cell subset expressing homing receptor L-selectin.
Authors: Tsuji T , Nibu R , Iwai K , Kanegane H , Yachie A , Seki H , Miyawaki T , Taniguchi N
Published: December 2022

The humoral response in newborns is mainly restricted to IgM production, which may be attributable to the naive nature of both B and T cells at birth. In light of the current evidence that memory (CD45RO+) CD4+ T cells help B cell differentiation, the present study was undertaken to examine whether a specified population within memory CD4+ T cells could induce the maturation of neonatal naive B cells. In the conventional PWM-stimulated cultures, the generation of IgG- and IgA-producing cells in addition to IgM production by neonatal B cells was significantly enhanced by co-cultures with memory, but not naive, CD4+ T cells. Memory CD4+ T cells were further divided into two populations based on expression of homing receptor L-selectin. These memory CD4+ T cell subpopulations appeared to behave in different fashions concerning help for Ig production by naive (sIgD+) and mature (sIgD-) B cells. L-selectin-negative memory CD4+ T cells exhibited helper function for Ig secretion by mature B cells. Intriguingly, Ig production by neonatal B cells as well as adult naive B cells, although less than that by mature B cells, was efficiently promoted by L-selectin-positive memory CD4+ T cells rather than L-selectin-negative ones. The results suggest that the capability of neonatal naive B cells to secrete IgG and IgA can be elicited by appropriate T-cell signals, especially from the L-selectin-positive population within memory CD4+ T cells, seemingly indicating its possible role for isotype switching in B cells.
Simultaneous regulation of CD2 adhesion and signaling functions by a novel CD2 monoclonal antibody.
Authors: van Kemenade F J , Tellegen E , Maurice M M , Lankester A C , Kuijpers T W , Brouwer M , de Jong R , Miedema F , van Lier R A
Published: December 2022

Accessory molecules on T cells can support adhesion and transduce agonistic signals that facilitate Ag receptor-induced T cell activation. The T cell differentiation Ag CD2 may exert both functions, as has been amply demonstrated in studies with CD2 mAbs. In addition, experiments in which either purified ligand (CD58) or transfected CD2 and CD58 molecules were used have confirmed this notion. However, controversy exists as to whether CD2 alters its affinity for CD58 in the course of T cell stimulation, and whether this putative affinity change affects CD2-mediated activation signals. We now describe a CD2 mAb (HIK27) that recognizes an epitope constitutively expressed on resting T cells and induces increased adhesiveness of CD2 toward CD58. Addition of HIK27 to a stimulatory but nonmitogenic pair of CD2 mAbs induces a strong proliferative response. Finally, HIK27 was found to be co-mitogenic with CD58 expressed on sheep erythrocytes, B cell lines, and CD58-transfected L cells. The simultaneous modulation of CD2 adhesion and signaling on HIK27 binding suggests that both functions of the molecule may be enhanced in the course of T cell stimulation.
A disease-related epitope of Torpedo acetylcholine receptor. Residues involved in I-Ab binding, self-nonself discrimination, and TCR antagonism.
Authors: Wall K A , Hu J Y , Currier P , Southwood S , Sette A , Infante A J
Published: December 2022

Residues 146-162 of the Torpedo californica acetylcholine receptor alpha-subunit contain the immunodominant T cell epitope for experimental autoimmune myasthenia gravis-susceptible C57BL/6 mice. To develop potential therapeutic peptides, a detailed analysis of the epitope was undertaken. Truncated and substituted synthetic peptides were tested as stimulators of T cell clones and immune lymph node cells. Critical residues spanned positions 151-159. Y151 and V156 were critical for MHC binding. The results indicated a general motif for binding to I-Ab to be an aromatic or hydrophobic residue (preferentially Y or F) at position i followed by an uncharged residue at position (i + 5). Lysine was not tolerated at position (i + 8). Residues D152, K155, S157, and I158 were important T cell contact residues. A peptide corresponding to the murine 146-162 sequence, which differs from the Torpedo sequence at 5 residues, bound to I-Ab but was nonimmunogenic, consistent with the assigned TCR and I-Ab contact residues. These results suggest that tolerance is responsible for the lack of T cell cross-reactivity with the murine acetylcholine receptor. Substituted peptides were tested for the inhibition of T cell clone responses and for TCR antagonism. Although peptides substituted at residues 157 and 158 inhibited most clones, no single peptide could completely inhibit all clones or primed lymph node cells. These findings indicate that antagonist peptides may be useful in inhibiting T cell responses to complex Ag displaying a single immunodominant epitope. Multiple antagonists used in combination may be required for maximum inhibition of the response.
The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b. Activation by a heterologous beta subunit and localization of a ligand recognition site to the I domain.
Authors: Bilsland C A , Diamond M S , Springer T A
Published: December 2022

p150,95 is a member of the leukocyte integrin family of adhesion proteins. Compared with LFA-1 and Mac-1, p150,95 is less well functionally characterized. Although p150,95 has complement receptor activity for iC3b and has been designated complement receptor type 4, transfected cells expressing p150,95 do not bind iC3b-sensitized cells. We report that cells cotransfected with a human p150,95 alpha subunit and a chicken, but not human, beta subunit bind IgM-iC3b-coated erythrocytes, suggesting that interactions between the alpha and beta subunits can regulate p150,95 adhesiveness. Furthermore, purified human p150,95 binds to cell-bound iC3b-coated erythrocytes. Because binding to iC3b by cellular and purified p150,95 is specifically abolished by mAbs that localize to the I domain of p150,95, we suggest that the I domain of the p150,95 alpha subunit is an important ligand recognition site for iC3b.
Eosinophil transendothelial migration induced by cytokines. II. Potentiation of eosinophil transendothelial migration by eosinophil-active cytokines.
Authors: Ebisawa M , Liu M C , Yamada T , Kato M , Lichtenstein L M , Bochner B S , Schleimer R P
Published: December 2022

Activation of HUVEC monolayers by IL-1 beta or TNF-alpha induces migration of eosinophils across the endothelial monolayer (i.e., transendothelial migration) in vitro. In the present study, we demonstrate that culture of freshly isolated eosinophils in the presence of IL-5 for 24 to 48 h before use in the assay dramatically potentiated CD18-dependent eosinophil transendothelial migration through unstimulated endothelial monolayers. Granulocyte macrophage (GM)-CSF induced eosinophil transendothelial migration but did not induce neutrophil transendothelial migration. When IL-1 beta-activated endothelial cells were used, GM-CSF, IL-3, or IL-5 caused only modest potentiation of eosinophil transendothelial migration. Since activated endothelial cells are known to produce GM-CSF, we hypothesized that endothelial-derived GM-CSF might play a role in the process of IL-1 beta-induced eosinophil transendothelial migration. IL-1 beta-activated endothelial monolayers grown on the permeable supports produced 0.3 +/- 0.1 ng/ml of GM-CSF during a 4-h incubation and neutralizing Ab against GM-CSF inhibited eosinophil transendothelial migration by 48%, suggesting that endothelial-derived GM-CSF may participate in the response. Eosinophils purified from bronchoalveolar lavage 18 to 20 h after experimental Ag challenge in the lungs of allergic volunteers showed enhanced transendothelial migration, indicating that the cells may have undergone cytokine activation in vivo. Eosinophil-active cytokines may contribute to the preferential accumulation of eosinophils in vivo in part via potentiation of eosinophil transendothelial migration.
Action and target cell specificity of human macrophage-stimulating protein (MSP).
Authors: Skeel A , Leonard E J
Published: December 2022

Macrophage-stimulating protein (MSP) induces mouse resident peritoneal macrophages to become responsive to the chemoattractant C5a and to ingest C3bi-coated erythrocytes. We now show that MSP action is not limited to complement-induced responses, because it also induced responsiveness to the noncomplement chemoattractant casein. In addition to stimulating responsiveness to attractants, MSP functioned alone as a chemoattractant for resident peritoneal macrophages, with an optimal concentration of approximately 0.2 nM. A critical difference between MSP and C5a is that resident macrophages did not migrate to C5a without an additional stimulus such as MSP in the cell suspension, whereas macrophages suspended in medium alone migrated to MSP in the attractant well. Thus, in contrast to C5a, MSP seems capable of a dual role, both activator and attractant. MSP had no effect on responsiveness of mouse peritoneal exudate macrophages to C5a; nor could it attract exudate macrophages or human blood monocytes. Absorption studies showed that resident macrophages have a receptor for MSP, but exudate macrophages do not. In view of these findings, it seems that the biological role of MSP is not as a recruiter of blood monocytes to sites of inflammation, but as an activator of mature macrophages. The MSP-induced activated state for responsiveness to C5a or C3bi was transient, and decayed at a first order rate with a t 1/2 of approximately 1 h. This is a new example of the transience of activation induced in macrophages by proinflammatory stimuli.
Physical association of complement receptor type 3 and urokinase-type plasminogen activator receptor in neutrophil membranes.
Authors: Xue W , Kindzelskii A L , Todd R F , Petty H R
Published: December 2022

A previous study has shown that Fc gamma RIIIB (CD16), an extensively glycosylated glycosyl-phosphatidylinositol-linked neutrophil membrane protein, specifically co-caps with the iC3b R (CR3; CD11b/CD18). This study tests the possible physical interactions of another extensively glycosylated glycosyl-phosphatidylinositol-linked protein, the urokinase-type plasminogen activator receptor (uPAR), with CR3. Receptors were labeled using fluorochrome-conjugated F(ab')2 fragments of an anti-CR3 mAb. In some cases cells were capped using second step F(ab')2 fragments of an anti-mouse F(ab')2 antiserum. After 30 min at 37 degrees C, 65 +/- 4% of the cells exhibited CR3 caps whereas 61 +/- 2% demonstrated uPAR caps. When CR3-capped cells were probed with F(ab')2 fragments of anti-uPAR conjugated to a distinct fluorochrome, 45 +/- 3% of the cells co-capped uPAR. When uPAR was capped, 48 +/- 2% of the cells co-capped CR3. Similar levels of co-capping were observed using a DNP-conjugated anti-CR3 F(ab')2 and an anti-DNP second step F(ab')2 reagent for capping or using FITC-uPA as a probing reagent. Furthermore, CR3-uPAR co-capping and/or co-clustering was also observed using anti-CR3 IgM and Mn2+ as integrin aggregation stimuli. Significant co-capping of anti-CD14, anti-CD59, anti-Mo5, anti-HLA, or NBD-PE (a lipid probe) was not observed. Moreover, CR3 and uPAR co-capping was blocked by N-acetyl-D-glucosamine, but not by six other saccharides, suggesting that a lectin-like site may participate in co-capping. This suggests that CR3 may regulate adhesive events by several mechanisms, including the regulation of the spatial distribution of uPAR.
Serum IgA-mediated neutralization of HIV type 1.
Authors: Burnett P R , VanCott T C , Polonis V R , Redfield R R , Birx D L
Published: December 2022

The sera of 33 HIV-1-infected individuals, previously shown to neutralize HIV-1MN in vitro, were screened by ELISA for IgA reactivity against rgp120MN and a synthetic V3MN loop peptide. Six were selected for evaluation of the effect of serum IgA from infected individuals on the in vitro infection of susceptible target cells by HIV-1MN. By using protein G immobilized on Sepharose, we depleted the sera of IgG to a level undetectable by nephelometry and viral envelope-specific ELISA. The IgA component of the IgG-depleted serum was affinity purified with immobilized jacalin, a lectin that selectively binds the IgA1 fraction of human Ig. IgG-depleted sera and purified IgA1 serum fractions showing IgA reactivity against rgp120MN and V3MN by ELISA inhibited the in vitro infection of CEM-ss cells by HIV-1MN, but sera depleted of both IgG and IgA1 did not. These data show that, like serum IgG, serum IgA from selected HIV-1-infected individuals is capable of neutralizing HIV-1MN in vitro. The biologic significance of this observation and the identities of serum IgA-recognized HIV-1 neutralization epitopes remain to be determined.
Target organ-specific up-regulation of the MRC OX-40 marker and selective production of Th1 lymphokine mRNA by encephalitogenic T helper cells isolated from the spinal cord of rats with experimental autoimmune encephalomyelitis.
Authors: Weinberg A D , Wallin J J , Jones R E , Sullivan T J , Bourdette D N , Vandenbark A A , Offner H
Published: December 2022

Lewis x Buffalo F1 rat lymphocytes express both forms of the allelic marker RT7.1 (Lewis) and RT7.2 (Buffalo). We generated myelin basic protein (MBP)-specific encephalitogenic F1 T helper cell lines and adoptively transferred them into naive irradiated Lewis recipients, which enabled us to detect and isolate donor T cells (with RT7.2) within the recipients. The spinal cord and cerebrospinal fluid (CSF) were highly enriched for the donor T cells compared with the blood and spleen. The donor cell number peaked on the first day of disease in the spinal cord and CSF and decreased as the disease progressed. A high percentage of the donor T cells isolated from the spinal cord were positive for the T helper cell activation marker OX-40, whereas a (lower) percentage of CSF donor cells expressed OX-40. Donor cells isolated from blood or spleen were negative for OX-40 expression. In contrast, the IL-2 receptor (CD25) was positive on all the transferred T cells in all tissue sites examined. Cell-sorting experiments showed that the MBP-specific donor cells were enriched for IFN-gamma, IL-2, TNF-alpha, and IL-3 mRNA when compared with the host-recruited spinal cord cells, whereas similar amounts of IL-10 mRNA were produced by both populations. Lymphokine mRNA production was also enriched in donor T cells isolated from the spinal cord compared with donor T cells isolated from the spleen. The spinal cord donor cells produced higher levels of IL-2, IFN-gamma, and IL-3 mRNA, whereas similar amounts of IL-10 and TNF-alpha mRNA were produced from donor cells isolated from the spleen and the spinal cord. Our data suggest that the amount/percentage, activation state, and enhanced lymphokine production at the site of inflammation are all important factors in determining the autoimmune potential of Ag-specific effector T helper cells.
Hyaluronate is costimulatory for human T cell effector functions and binds to CD44 on activated T cells.
Authors: Galandrini R , Galluzzo E , Albi N , Grossi C E , Velardi A
Published: December 2022

Lymphohematopoiesis, cell matrix adhesion, homing of leukocytes, T cell activation, and tumor metastasis are mediated through the CD44 family of cell surface receptors. We have recently shown that anti-CD44 mAb trigger protein tyrosine kinase-dependent activation of T cell effector functions. Here, we show that hyaluronate (HA), a CD44 ligand, in conjunction with CD3/TCR-mediated stimuli, is costimulatory for human peripheral blood T cell proliferation, for IL-2 production by Th clones, and for release of trypsin-like esterase by cytolytic T cell clones. A human T cell line, HUT-78, was found to bind HA and on HA coating it was used as a target for cytolytic T cell clones. After anti-CD3 stimulation, CD3+/CD8+ clones acquire the ability of lysing HA-coated HUT-78 cells more efficiently than the same HA-uncoated targets. Resting peripheral blood T cells and T cell clones do not adhere to HA-coated plates. However, 24-h anti-CD3 mAb stimulation gives them the transient ability to bind HA. HA adhesion of activated T cells and T cell clones, as well as that of T cell lines, is blocked by one anti-CD44 mAb (J-173). Two other anti-CD44 mAbs induce a 10-fold increase in HA adhesiveness of anti-CD3-stimulated peripheral blood T cells. This impressive HA adhesiveness is also readily blocked by J-173 anti-CD44 mAb. These data indicate that 1) HA is costimulatory for human T cell effector functions in conjunction with CD3/TCR-mediated stimuli, 2) the capacity to bind HA is acquired by resting T cells and T cell clones after anti-CD3 stimulation, and 3) HA binding occurs via specific interaction with CD44 molecules expressed on activated T cells.
Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by pro-inflammatory cytokines.
Authors: Cubitt C L , Lausch R N , Oakes J E
Published: December 2022

Neutrophils and Langerhans cells participate in inflammatory reactions within the human cornea. Because granulocyte-macrophage (GM)-CSF is a chemotactic and activating factor for these two cell types, we investigated whether this cytokine is produced by human corneal epithelial cells and corneal fibroblasts. Cultures of each cell type were exposed to increasing concentrations of IL-1 alpha or TNF-alpha. Culture supernatants were assayed for GM-CSF by using ELISA and cytokine mRNA levels were monitored by using reverse transcriptase-PCR. IL-1 alpha treatment of both cell types resulted in the appearance of GM-CSF mRNA and the production of > 480 pg protein/10(6) cells. However, TNF-alpha treatment yielded divergent results. Stimulation of epithelial cells with TNF-alpha resulted in the appearance of > 560 GM-CSF mRNA molecules per cell and production of > 1300 pg GM-CSF/10(6) cells. In contrast, stimulation of corneal fibroblasts resulted in < 16 GM-CSF mRNA molecules/cell and < 60 pg GM-CSF/10(6) cells. Binding studies with 125I-labeled TNF-alpha revealed that corneal fibroblasts had as many receptor sites as did corneal epithelial cells. Furthermore, corneal fibroblasts could respond to TNF-alpha-receptor-mediated signal transduction because they produced nanogram amounts of IL-6 after being treated with this cytokine. The results suggest that both cell types synthesize GM-CSF in response to IL-1 alpha, but that only corneal epithelial cells produce significant amounts of GM-CSF after TNF-alpha exposure. Differences in the responses of the two cell types to TNF-alpha may reflect a means of limiting accumulation of neutrophils and Langerhans cells and, thus, minimize corneal damage.
Lipolysaccharide-induced monocyte retention in the lung. Role of monocyte stiffness, actin assembly, and CD18-dependent adherence.
Authors: Doherty D E , Downey G P , Schwab B , Elson E , Worthen G S
Published: December 2022

Blood monocytes and monocyte-derived macrophages accumulate in the lungs and can modulate pulmonary inflammatory and reparative processes through their elaboration of cytokines and growth factors. Endotoxemia, often a prelude to acute lung injury, induces a monocytopenia, likely resulting from monocyte accumulation in the lung. We hypothesized that LPS would induce monocyte lung retention by increasing monocyte stiffness and thereby diminishing the cell's ability to deform and transit the narrow pulmonary capillary network, and that LPS would induce CD18-dependent adhesion of monocytes to endothelium, prolonging their retention. LPS induced a rapid and concentration-dependent increase in human monocyte stiffness, net filamentous actin assembly, and retention in a filtration model of pulmonary capillaries. These LPS-induced responses were dependent on the integrity of actin filaments in that cytochalasin D, an agent that disrupts filamentous actin assembly, attenuated each of these processes. LPS induced CD18-dependent and -independent human monocyte adhesion to unstimulated human endothelial cell monolayers. In vivo, rabbit monocytes were retained in the lungs of animals rendered endotoxemic. Pretreatment of monocytes ex vivo with LPS enhanced their lung retention suggesting that LPS was acting directly on monocytes. Initial lung retention during endotoxemia was attenuated by inhibiting monocyte F-actin assembly with cytochalasin D. Anti-CD18 Abs caused a slight decrease in initial retention of monocytes, but led to a 90% inhibition of retention by 2 h. Control IgG had no effect. These data suggest that the initial retention of monocytes in the lung during endotoxemia is dependent on alterations in their stiffness and assembly/organization of F-actin, and that CD18-dependent adhesive mechanisms prolong monocyte retention in the lung during this process.
Regulation of C1q receptor expression on human polymorphonuclear leukocytes.
Authors: Jack R M , Lowenstein B A , Nicholson-Weller A
Published: December 2022

The C1q receptor (C1qR) is expressed on a variety of cells, including polymorphonuclear leukocytes (PMN), in which stimulation by the C1qR leads to activation as measured by superoxide production. To investigate the expression and modulation of the C1qR on PMN, the binding of biotinylated C1q to PMN in suspension was measured by flow cytometry. Biotinylated C1q bound in a saturable and specific manner to PMN and the use of low ionic strength buffers enhanced binding. Covalent coupling of C1q to Sepharose beads allowed the affinity precipitation of a single 125-kDa band from surface iodinated PMN. The apparent molecular mass of the C1qR increased to 135 kDa upon reduction. Freshly isolated PMN had a uniform expression of C1qRs and phorbol myristate acetate induced a unimodal up-regulation of receptors. The inflammatory peptide FMLP rapidly up-regulated receptors by up to fivefold, and the high level of expression remained constant over 45 min. Taxol inhibited the FMLP induction of C1qR up-regulation, indicating that the ability to move the intracellular stores of C1qR depends on normal microtubule functioning. Thus, the C1qR is a constitutively expressed protein of the human PMN plasma membrane and it is rapidly up-regulated from a discrete intracellular pool of preformed molecules with the same kinetics as CR1 and CR3. It is likely that the C1qR is a component of the PMN complement receptor exocytic vesicle (CREV), in which CR1 and CR3 are also stored.
IL-7 stimulates CSF-induced proliferation of murine bone marrow macrophages and Mac-1+ myeloid progenitors in vitro.
Authors: Jacobsen F W , Veiby O P , Jacobsen S E
Published: December 2022

The role of IL-7 as an important stimulator of the growth of B and T cell precursors, as well as mature T cells, is well established. In contrast, the role of IL-7 in myelopoiesis has not been characterized thoroughly, and thus, IL-7 has been regarded as a lymphoid lineage-restricted cytokine. However, we have recently reported that IL-7 enhanced CSF-induced myeloid proliferation of primitive murine hematopoietic (Lin-Sca-1+) progenitors, whereas IL-7 did not affect significantly the proliferation of a population of more mature (Lin-) progenitors. The present study was initiated to investigate further whether IL-7 might affect CSF-induced proliferation of subpopulations of committed myeloid progenitors as well as mature bone marrow macrophages. IL-7 enhanced macrophage colony-stimulating factor (CSF-1)-induced colony formation of single bone marrow macrophages 90%, whereas IL-7 alone had no effect. Furthermore, IL-7, in a concentration-dependent manner, increased the proliferation of mononuclear cells expressing the Mac-1 Ag (Mac-1+ mononuclear cells (MNC); CD11b) up to fivefold in response to CSF-1, granulocyte macrophage-CSF (GM-CSF), or IL-3. In contrast, no effect of IL-7 was observed on Mac-1- MNC. The synergistic effect of IL-7 on Mac-1+ MNC was caused by an increase in macrophage colonies (CFU-M) and mixed granulocyte-macrophage colonies (CFU-GM), whereas the total number of granulocyte colonies (CFU-G) was not affected. This suggests that IL-7 can provide proliferative signals to Mac-1+ progenitors with a macrophage potential, but not to progenitors committed to pure granulocyte differentiation.