bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of HIV-1 envelope-specific CD4+ T cell lines simultaneously established from peripheral blood mononuclear cells and lymph node biopsy in HIV-1-infected individuals.
Authors: Ratto-Kim S , Sitz K V , Scherer A M , Kim J H , Anderson D W , Nau M E , Mann D L , Akolkar P N , Gulwani-Akolkar B , Silver J , Birx D L
Published: December 2022

HIV-1 envelope-specific CD4+ T cell lines were established simultaneously from PBMC and lymph node mononuclear cells of two HIV-1-infected patients. Three recombinant envelope proteins were used to establish the CD4+ T cell lines: gp160NL4-3, gp120IIIB, and gp120MN. Six T cell lines were established from the first patient, one for each Ag from each compartment, and four T cell lines, two per compartment, were established from the second patient. Each line was challenged with a panel of overlapping peptides spanning the entire gp120 sequence to define its T cell epitope specificity. The pattern of recognition for all the lines from any given patient was similar between compartments. Each patient had a different pattern of peptide recognition. TCR analysis showed a heterogeneous usage of Vbeta between lines with same peptide specificity and established from different compartments. These data suggest that the cellular immune response does not phenotypically vary between the peripheral blood and lymph node compartments, but demonstrates genotypic heterogeneity, showing possible redundancy of the immune response to HIV-1 gp160.
Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity.
Authors: Niizeki H , Alard P , Streilein J W
Published: December 2022

Since nerve termini on Langerhans cells (LC) contain calcitonin gene-related peptide (CGRP), and since ultraviolet B radiation (UVR) causes CGRP to be released from cutaneous nerve endings, we examined whether CGRP participates in the immune aberrations caused in skin by UVR. First, intradermally injected CGRP, in a dose-dependent manner, reduced LC density and impaired CH induction when hapten was painted on the injected site. Second, CGRP antagonist restored CH induction after UVR. Third, anti-TNF-alpha Abs injected before CGRP prevented the loss of LC density and restored CH induction. Fourth, CGRP failed to impair CH induction in mast cell-deficient mice. Fifth, CGRP induced mast cells to release TNF-alpha. We conclude that CGRP plays an essential role in the loss of CH induction after UVR. These data indicate that UVR, by causing the release of CGRP from cutaneous nerve endings, triggers mast cell release of TNF-alpha, which impairs CH induction.
Induction of HLA-A2-restricted CTLs to the mucin 1 human breast cancer antigen.
Authors: Apostolopoulos V , Karanikas V , Haurum J S , McKenzie I F
Published: December 2022

HLA-A*0201/Kb transgenic mice were immunized with oxidized mannan-mucin 1 (MUC1) as a fusion protein (containing five repeats of the 20-amino-acid MUC1 VNTR (variable number of tandem repeats) that generated highly active CD8+ CTLs to MUC1 peptides. In a direct CTL assay, the MUC1 peptides could be presented specifically by both the transgenic murine HLA-A*0201/Kb and human HLA-A*0201 molecules. The 9-mer MUC1 peptide sequences (APDTRPA and STAPPAHGV) were presented by HLA-A*0201, although they did not contain L at P2 and L/V at P9, the preferred motifs; as a consequence, the binding was of relatively low affinity when compared with a high affinity-binding HIV peptide (ILKEPVHGV). In addition, when mice were immunized separately with the HLA-A*0201-binding peptides (STAPPAHGV or APDTRPAP-containing peptides-keyhole limpet hemocyanin-mannan), direct lysis of MCF-7 (HLA-A*0201+, MUC1+) also occurred. The findings are of interest for tumor immunotherapy, particularly as the CTLs generated to low affinity-binding peptides were highly active and could specifically lyse an HLA-A*0201+ human breast cancer cell line without further in vitro stimulation. The findings demonstrate that the range of peptides that can generate CTLs is broader than formerly considered.
Apoptotic cell death upon contact of CD4+ T lymphocytes with HIV glycoprotein-expressing cells is mediated by caspases but bypasses CD95 (Fas/Apo-1) and TNF receptor 1.
Authors: Ohnimus H , Heinkelein M , Jassoy C
Published: December 2022

Loss of CD4+ T helper lymphocytes is central to the development of immunodeficiency after infection with HIV. In this study, we demonstrate that contact of primary uninfected CD4+ T lymphocytes with HIV-infected or HIV envelope glycoprotein-expressing cells results in apoptotic cell death of both uninfected and infected cells. Apoptosis was blocked by inhibitors of caspases/IL-1beta-converting enzyme-like proteases. This finding provides conclusive evidence that cytotoxicity upon contact of HIV-infected and uninfected primary cells is an active process and represents another example for the role of caspases in the induction of apoptosis. Prevention of apoptosis by inhibition of caspases did not block the formation of syncytia, indicating that apoptosis occurs either in a subpopulation of cells or in syncytia. Cell death was not mediated by the CD95 (Fas/Apo-1) or TNF receptor 1 molecules, which indicates a different pathway of apoptosis induction. The data indicate that initiation of apoptosis significantly shortens the life span of uninfected CD4+ T cells upon contact with HIV-infected cells and may represent a factor that contributes to the destruction of CD4+ T lymphocytes in vitro. Elucidation of the mechanism that initiates apoptosis in this situation will add to our understanding of both HIV pathogenesis and apoptotic signaling.
Identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate T cells.
Authors: Howard C J , Sopp P , Brownlie J , Kwong L S , Parsons K R , Taylor G
Published: December 2022

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.
Evidence for multiple promoters of the human IL-5 receptor alpha subunit gene: a novel 6-base pair element determines cell-specific promoter function.
Authors: Zhang J , Kuvelkar R , Cheewatrakoolpong B , Williams S , Egan R W , Billah M M
Published: December 2022

In addition to a previously characterized promoter (P1), we now show the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nuclease protection assays. Transfection of eosinophilic HL-60 cells with reporter gene constructs in which either P1 or P2 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in CAT expression; little or no CAT expression occurred in other myeloid and nonmyeloid cell lines. Deletion studies showed that a 66-bp region, ranging from -31 to +35, was sufficient to promote CAT expression in eosinophilic HL-60 cells. Analysis of linker-scanning mutants identified a novel 6-bp element (5' CTAATT 3') spanning -19 to -14 that was essential for P2 promoter activity. In electrophoretic mobility shift assays, a P2 region from -31 to +1 containing the unique 6-bp element, when used as a probe, formed a complex with a protein(s) that was found only in the eosinophilic cell line. This binding activity was lost upon replacement of the 6-bp element with a 6-bp linker, suggesting that this element likely serves as the binding site for an eosinophilic HL-60 cell-specific transcription factor(s). Together, these data suggest an important role for P2 promoter in the regulation of eosinophil-specific expression of the human IL-5 receptor alpha gene.
IL-4-induced STAT6 suppresses IFN-gamma-stimulated STAT1-dependent transcription in mouse macrophages.
Authors: Ohmori Y , Hamilton T A
Published: December 2022

IL-4 suppresses the IFN-gamma-induced expression of the IFN regulatory factor-1 (IRF-1) gene, and this suppression is attenuated by increasing the amount of IFN-gamma. The effects of IFN-gamma and IL-4 on transcription of a reporter gene under control of a 1.3-kb fragment from the IRF-1 gene promoter or the STAT binding element (SBE) from this gene in the context of a heterologous promoter are similar to their effects on the endogenous IRF-1 gene. IFN-gamma-dependent transcription of reporter gene is suppressed by IL-4, but IL-4 alone has no trans-activating function. IL-4 treatment does not inhibit the tyrosine phosphorylation or nuclear translocation of IFN-gamma-activated STAT1. Rather, IFN-gamma and IL-4 independently activate STAT1 and STAT6, respectively, and both proteins bind to the IRF-1 SBE in homodimeric form. The affinity of STAT1 for the IRF-1 SBE is higher than the affinity of STAT6, as measured by competition with unlabeled oligonucleotide. These observations suggest that IL-4 may suppress IFN-gamma-stimulated transcription of the IRF-1 gene by activation of STAT6, which can compete with STAT1 for occupancy of the IRF-1 SBE when STAT1 levels are low. Suppression may be attenuated as the quantity of STAT1 relative to that of STAT6 increases in cells treated with increasing amounts of IFN-gamma and displaces STAT6.
IL-18 protects mice against pulmonary and disseminated infection with Cryptococcus neoformans by inducing IFN-gamma production.
Authors: Kawakami K , Qureshi M H , Zhang T , Okamura H , Kurimoto M , Saito A
Published: December 2022

We examined the effects of a newly identified cytokine, IL-18, originally designated as IFN-gamma-inducing factor, in a mouse model of pulmonary and disseminated infection with a highly virulent strain of Cryptococcus neoformans. Administration of murine rIL-18 enhanced elimination of live micro-organisms from the lungs, prevented fungal dissemination to the brain, reduced the level of serum cryptococcal capsular polysaccharide Ags, and increased the survival rate of infected mice. Histologic examination of lung sections of infected and PBS-treated mice showed a poor cellular inflammatory reaction and a large number of multiplying C. neoformans yeast cells in alveolar spaces. In contrast, massive infiltration of inflammatory cells, consisting mainly of mononuclear cells, characterized sections of lungs of infected animals treated with IL-18. Treatment with IL-18 also increased the level of serum IFN-gamma. In addition, the protective effect of IL-18 on cryptococcal infection was abrogated by administration of neutralizing anti-IFN-gamma mAb. Finally, we examined the effect of neutralizing anti-IL-18 Ab on cryptococcal infection to define the physiologic role of this cytokine in host defense using another weakly virulent strain of C. neoformans, which induced the expression of IL-18 mRNA in the infected lungs. Administration of this Ab exacerbated the infection, as shown by the increased lung burden. Our results indicate that IL-18 plays an important role in host defense against infection with C. neoformans.
Interchangeable endotoxin-binding domains in proteins with opposite lipopolysaccharide-dependent activities.
Authors: Schumann R R , Lamping N , Hoess A
Published: December 2022

Host defense against microorganisms involves proteins that bind specifically to bacterial endotoxins (LPS), causing different cellular effects. Although LPS-binding protein (LBP) can enhance LPS activities, while bactericidal/permeability-increasing protein (BPI) and Limulus anti-LPS factor (LALF) neutralize LPS, it has been proposed that their LPS-binding domains possess a similar structure. Here, we provide evidence that the LBP/LPS-binding domain is, as in the LALF structure, solvent exposed and therefore available for LPS binding. Our investigations into the activity of LPS-binding domains of different LPS-binding proteins, in the context of LBP, provide the first functional analysis of these domains in a whole protein. We constructed domain exchange hybrid proteins by substituting 12 amino acids of the LBP/LPS-binding domain with those of BPI and LALF and expressed them in Chinese hamster ovary cells. Although discrete point mutations within the LPS-binding domain of LBP disrupted its specific functions, the hybrid proteins were still able to bind LPS and, in addition, retained the wild-type LBP activity of enhancing LPS priming for FMLP-induced oxygen radical production by neutrophils and transferring LPS aggregates to CD14. Although BPI and LALF display opposite activities to LBP, and LALF does not share any sequence homology with LBP, our data provide strong evidence that LBP, BPI, and LALF possess a solvent-exposed, interchangeable LPS binding motif that is functionally independent of LPS transport or neutralization.
Human alternative complement pathway-mediated lysis of rabbit erythrocytes is enhanced by natural anti-Galalpha1-3Gal antibodies.
Authors: Tomlinson S , Nussenzweig V
Published: December 2022

Human serum is effective at lysing unsensitized rabbit erythrocytes due to activation of the alternative complement pathway. Hemolysis is known to be enhanced by the addition of human IgG. We demonstrate that the enhancement of alternative pathway-mediated rabbit E lysis by human Ig is due to natural Abs specific for galactosyl (Gal)alpha1-3Gal. Depletion of normal human serum (NHS) of anti-Galalpha1-3Gal Abs removes almost all hemolytic activity from NHS, and activity can be restored by repletion with anti-Galalpha1-3Gal affinity-purified Abs. Factor B-depleted serum (inactive alternative pathway) did not lyse rabbit erythrocytes. Although C2-depleted serum was fully hemolytic, activity was removed by depletion of anti-Galalpha1-3Gal Abs. Of the total anti-Galalpha1-3Gal Abs affinity isolated from NHS, about one-third were IgM and two-thirds were IgG. Both anti-Galalpha1-3Gal IgG and IgM enhanced alternative pathway-mediated erythrocyte lysis.
Do effector and memory T helper cells also need B7 ligand costimulatory signals?
Authors: Gause W C , Mitro V , Via C , Linsley P , Urban J F , Greenwald R J
Published: December 2022

Blocking B7 ligand-costimulatory molecules can inhibit a primary T-dependent immune response, but whether these interactions also mediate ongoing or memory immune responses is less clear. Development of immunotherapies based on blocking B7 ligand interactions would be limited if they were effective only at the initiation of an immune response. We discuss the conditions under which T helper effector and memory cells may or may not require B7 ligand interactions for their function.
Inhibition of acute peritoneal inflammation in rats by a cytokine-induced neutrophil chemoattractant receptor antagonist.
Authors: Zagorski J , Wahl S M
Published: December 2022

Cytokine-induced neutrophil chemoattractant (CINC), a member of the IL-8 superfamily of chemokines, is one rat homologue of the three human GRO proteins. Neutralizing Abs against CINC have been shown previously to be efficacious in several models of inflammation, indicating that CINC is an important proinflammatory mediator in vivo. By introducing the N-terminal mutation delta1-5,ELR>AAR into CINC, we have developed a rat alpha-chemokine receptor antagonist (ra) analogous to a previously described mutant of human IL-8. Bacterially expressed CINCra had no chemotactic activity itself, but completely blocked the activity of physiologic concentrations of CINC when used as an antagonist in vitro. Inhibition by CINCra was specific, since it had approximately 10-fold less antagonist activity on the related, but distinct rat alpha-chemokine macrophage-inflammatory protein 2. When coinjected with zymosan into the peritoneal cavities of Lewis rats, 100 microg of CINCra inhibited neutrophil influx by approximately 40%, which was comparable with inhibition caused by polyclonal anti-CINC Abs.
B cell responses to a peptide epitope: III. Differential T helper cell thresholds in recruitment of B cell fine specificities.
Authors: Agarwal A , Rao K V
Published: December 2022

Th cell requirements in the individual stages comprising a murine humoral response to a synthetic peptide were examined. Induction of a T-dependent IgM response was readily achieved in the presence of unprimed or low numbers of Ag-primed T cells. In contrast, class switch to the IgG isotype of Abs demanded a markedly elevated frequency of primed T cells and occurred concomitantly with B cell differentiation into an membrane-bound IgG+ memory population. These results indicated that induction and progression of a T-dependent humoral IgG response were comprised of a single rate-limiting step represented by that involving Ab isotype switch. Subsequent studies established that this also represented the principal step where antibody-purifying mechanisms operate. This was enforced by imposing a threshold barrier for Th cell recruitment by early Ag-activated B cells to enable class switch and consequent retention as the response progresses. The quantum of this threshold, however, was not invariant, but, rather, was described as a balance between the affinity of B cell receptor for Ag and the frequency of Ag-specific Th cells.
ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.
Authors: Eischen C M , Williams B L , Zhang W , Samelson L E , Lynch D H , Abraham R T , Leibson P J
Published: December 2022

Activation-induced cell death (AICD) is initiated by the TCR-dependent up-regulation of Fas ligand (FasL) mRNA. The subsequently generated soluble or cell-associated FasL gene products bind Fas, leading to apoptosis of the T cells. Although TCR stimulation is essential to initiate AICD, little is known about which TCR-initiated second messengers are required for FasL expression. We provide evidence in this work that T cells lacking the tyrosine kinase ZAP-70 are unable to up-regulate FasL and undergo AICD. Transfection of wild-type ZAP-70 into the ZAP-70-deficient T cells restores their sensitivity to TCR-induced apoptosis, whereas transfection of catalytically inactive ZAP-70 does not. These results provide clear evidence that ZAP-70 tyrosine kinase is essential in up-regulating FasL for TCR-induced apoptosis.
RP1, a new member of the adenomatous polyposis coli-binding EB1-like gene family, is differentially expressed in activated T cells.
Authors: Renner C , Pfitzenmeier J P , Gerlach K , Held G , Ohnesorge S , Sahin U , Bauer S , Pfreundschuh M
Published: December 2022

Cross-linking of the CD3 and CD28 molecules on T lymphocytes represents one of the most effective signals for T lymphocyte activation and triggering of their cytotoxic effector function. To identify genes that are expressed in T cells after stimulation, mRNA from T lymphocytes that had been activated by the simultaneous stimulation of the CD3 and CD28 trigger molecules was transcribed for a differential mRNA display analysis into cDNA and was compared with cDNA from CD28- or CD3-activated or resting lymphocytes. Differential expression was confirmed subsequently by Northern blot analysis. One of the cDNA fragments expressed specifically in CD3- and CD28-activated T cells was designated RP1. The predictive protein-coding region of RP1 had a significant homology to members of the recently found adenomatous polyposis coli (APC) protein-binding EB1 gene family, which codes for yet unknown protein(s). Bacterially expressed RP1 protein revealed specific binding to wild-type but not to mutated APC protein. The rapid up-regulation of RP1 mRNA in properly activated T cells suggests that this gene might belong to the immediate/early gene family, which controls the signal transduction cascade downstream of the TCR. As the expression level of the RP1 gene in activated T cells and a spectrum of tumor-derived cell lines correlates with the proliferative status of the cells, members of the EB1-like gene family may not only be involved in the tumorigenesis of colorectal cancers but may also play a role in the proliferative control of normal cells.
Peptides derived from a wild-type murine proto-oncogene c-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts.
Authors: Nagata Y , Furugen R , Hiasa A , Ikeda H , Ohta N , Furukawa K , Nakamura H , Furukawa K , Kanematsu T , Shiku H
Published: December 2022

In this analysis, we examined whether peptides derived from a wild-type murine proto-oncogene, c-erbB-2, function as tumor rejection Ags. Expression of murine c-erbB-2 examined by means of reverse transcription-PCR was observed in several normal adult tissues, such as intestine, kidney, and testis. We then transduced human and murine c-erbB-2 cDNA into two mutually noncross-reactive fibrosarcoma lines of BALB/c origin, CMS7 and CMS17. In BALB/c mice immunized with CMS17HE (CMS17 transduced with human c-erbB-2 cDNA), the growth of subsequently challenged CMS7HE (CMS7 transduced with human c-erbB-2 cDNA) was significantly suppressed. CTL against human c-erbB-2-expressing cells were generated from BALB/c spleen cells in vivo and in vitro sensitized by CMS17HE. The CTL activity was also directed against murine c-erbB-2-expressing cells, CMS7ME and CMS17ME, and was blocked by anti-CD8 or anti-Kd mAbs. A series of peptides of human or murine c-erbB-2 compatible with the Kd binding motif was synthesized. The CTL were reactive with P1.HTR (H-2d) pulsed with three of these peptides, p63-71 (human c-erbB-2 derived), p63-71(A) (murine c-erbB-2 derived), and p780-788 (common for human and murine c-erbB-2). Spleen cells immunized in vivo and in vitro with syngeneic spleen cells pulsed with these peptides became cytotoxic for CMS17HE and/or CMS17ME, but not CMS17neo (CMS17 transduced with control vector). The growth of CMS7ME was suppressed in mice immunized with the murine c-erbB-2-derived peptide, p63-71(A) or p780-788. There was no apparent pathologic change in mice that rejected CMS7ME after vaccination with these peptides.
Binding of malaria T cell epitopes to DR and DQ molecules in vitro correlates with immunogenicity in vivo: identification of a universal T cell epitope in the Plasmodium falciparum circumsporozoite protein.
Authors: Calvo-Calle J M , Hammer J , Sinigaglia F , Clavijo P , Moya-Castro Z R , Nardin E H
Published: December 2022

The efficacy of a malaria peptide vaccine would be enhanced by the inclusion of a parasite-derived universal T cell epitope to ensure that all vaccinees develop parasite-specific cellular and humoral immunity. Two circumsporozoite (CS) protein T cell epitopes, previously identified by CD4+ T cell clones derived from Plasmodium falciparum sporozoite-immunized volunteers, were studied to determine their HLA class II binding potential. One epitope, located in amino acid (aa) 326-345 of the P. falciparum (NF54 strain) CS protein, was "universal" in that it could bind to multiple DR and DQ molecules in vitro. In contrast, the second epitope, T1, which is located in the CS repeat region, was recognized by T cells in the context of DQ6 (DQB1*0603) and did not bind with high affinity to any of the class II molecules tested in the peptide binding assays. The in vitro patterns of peptide/HLA interactions correlated with immunogenicity in vivo. A multiple antigen peptide (MAP) containing the aa 326-345 epitope elicited responses in eight inbred strains (H-2(a,b,d,k,p,q,r,s)), while the T1 MAP was recognized by only a single haplotype, H-2b. The combination of the universal aa 326-345 T cell epitope and the T1 repeat in a di-epitope MAP overcame the genetic restriction to the P. falciparum CS repeat region and elicited antisporozoite Ab responses in all of the MAP-immunized mice. Synthetic peptide malaria vaccines containing the aa 326-345 universal T cell epitope would be expected to elicit parasite-specific immune responses in both sporozoite-primed and naive individuals of diverse genetic backgrounds.
Time-dependent induction of protective anti-influenza immune responses in human peripheral blood lymphocyte/SCID mice.
Authors: Albert S E , McKerlie C , Pester A , Edgell B J , Carlyle J , Petric M , Chamberlain J W
Published: December 2022

The human (hu) PBL/SCID mouse model has the potential to provide a powerful tool for the study of human immune function. However, at peak engraftment (4-8 wk postinjection), recovered human T cells are largely unresponsive to foreign Ag and have converted to an activated/memory-type phenotype. Here we show that this conversion is not a prerequisite for engraftment because at early stages (2 wk) a substantial fraction of human T cells detected in SCID peripheral blood retains the unactivated/naive phenotype of donor PBL. This early stage is also associated with a TCR repertoire in both the CD4 and CD8 subsets that is similar to that in the donor. Importantly, we show that strong HLA class I allele- and peptide-specific cytotoxic T lymphocyte as well as humoral responses can be generated in this model when human cells encounter Ag (infection with influenza A) at early, but not late, stages in engraftment. This early human response was also functional, as partial protection against influenza-induced pathology and death in SCIDs was observed. Taken together, these results demonstrate that the huPBL/SCID model can support the generation of potent and specific CTL and humoral responses provided that Ag is introduced early, presumably before the time-dependent generalized xenoactivation of engrafted human cells.
The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis.
Authors: Keane M P , Arenberg D A , Lynch J P , Whyte R I , Iannettoni M D , Burdick M D , Wilke C A , Morris S B , Glass M C , DiGiovine B , Kunkel S L , Strieter R M
Published: December 2022

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder. Fibroplasia and deposition of extracellular matrix are dependent, in part, on angiogenesis. We postulated that an imbalance exists in the expression of angiogenic (IL-8) vs angiostatic (IFN-gamma-inducible protein (IP-10)) CXC chemokines, which favors net angiogenesis in IPF. To test this hypothesis, we obtained open lung biopsies either from normal patients undergoing thoracic surgery for reasons other than interstitial lung disease (control) or from patients with IPF. We found that levels of IL-8 were greater from tissue specimens of IPF patients then from those of controls. In contrast, IP-10 levels were higher from tissue specimens obtained from control subjects than from those from IPF patients. When IL-8 or IP-10 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced or enhanced, respectively. Immunolocalization of IL-8 demonstrated that the pulmonary fibroblast (PF) of IPF lung was the predominant cellular source of IL-8. Isolated PF from IPF patients constitutively produced more IL-8 and less IP-10 than control PF. Conditioned media from IPF-PFs demonstrated constitutive angiogenic activity that was attributable, in part, to IL-8. Depletion of IP-10 from IPF-PF CM resulted in an increase in corneal neovascularization. These findings support the notion that IL-8 and IP-10 are important factors that regulate angiogenic activity in IPF.
Nitric oxide inhibits IgE-mediated degranulation of mast cells and is the principal intermediate in IFN-gamma-induced suppression of exocytosis.
Authors: Eastmond N C , Banks E M , Coleman J W
Published: December 2022

IFN-gamma regulates various aspects of rodent peritoneal mast cell function, including mediator release, cell growth, TNF-alpha-mediated cytotoxicity, and MHC class II expression. We investigated whether the suppressive action of IFN-gamma on IgE/Ag-mediated degranulation of mast cells is mediated via synthesis of nitric oxide. Incubation of mouse peritoneal cells with L-NMMA, an inhibitor of nitric oxide synthase, or in medium lacking the nitric oxide precursor L-arginine reversed the inhibitory effect of IFN-gamma on Ag-induced serotonin release. Furthermore, the nitric oxide donors sodium nitroprusside and S-nitrosoglutathione inhibited degranulation, and this effect was direct, since it was seen equally on purified and unfractionated mast cells and occurred independently of IFN-gammaR expression. Additional experiments revealed that accessory cells in peritoneal cell populations were the principal target for the action of IFN-gamma and the main source of nitric oxide; the cytokine was more potent on unfractionated compared with purified mast cells, and IFN-gamma induced detectable nitrite production in mixed peritoneal cells, but not in purified mast cells. These studies show that IFN-gamma induces nitric oxide production in peritoneal cell populations, and that synthesized nitric oxide directly inhibits the IgE-mediated secretory function of mast cells. The activation of nitric oxide-producing cells in the tissue microenvironment may be important in the control of mast cell-dependent allergic reactions.
Eotaxin-induced eosinophil migration in the peritoneal cavity of ovalbumin-sensitized mice: mechanism of action.
Authors: Das A M , Flower R J , Perretti M
Published: December 2022

Cell accumulation in response to i.p. administration of the C-C chemokine, eotaxin, was studied in vivo. OVA-sensitized mice, exhibiting blood eosinophilia, had greater eosinophil (Eø) accumulation in response to 500 ng of eotaxin at 6 h (vehicle-injected, 3.0 +/- 0.5 x 10(5); eotaxin-injected, 8.6 +/- 1.0 x 10(5)) than nonsensitized, eotaxin-injected mice (2.5 +/- 0.4 x 10(5)). A nonspecific neutrophil migration was observed in both vehicle- and eotaxin-injected cavities. The number of intact mast cells in the peritoneal lavages after eotaxin injection was significantly lower than that in vehicle-injected animals (0.8 +/- 0.3 x 10(4) vs 2.8 +/-0.6 x 10(4), respectively). When endogenous peritoneal mast cells were depleted with compound 48/80 before eotaxin administration, there was a 51% reduction in Eø accumulation. This suggests an important role for endogenous mast cells in mediating the actions of eotaxin. The potential role of mast cell mediators in the actions of eotaxin was also investigated. Pretreatment with histamine-H1 or serotonin antagonists reduced Eø migration in response to eotaxin by 50 to 65%. Further, following pretreatment with a specific mAb against TNF-alpha, only nonspecific neutrophil influx was attenuated. Using neutralizing mAbs, Eø migration was found to be dependent on the adhesion molecules P- and E-selectin and CD11b. Eø accumulation was also sensitive to dexamethasone, with doses as low as 0.2 mg/kg inducing 100% inhibition. This study provides useful insight into the mechanisms of action of eotaxin.
Recombinant guinea pig and human RANTES activate macrophages but not eosinophils in the guinea pig.
Authors: Campbell E M , Proudfoot A E , Yoshimura T , Allet B , Wells T N , White A M , Westwick J , Watson M L
Published: December 2022

To characterize the biologic activities of potential mediators of allergic inflammation, we have cloned, expressed, and purified guinea pig RANTES (gpRANTES). cDNA for gpRANTES was cloned from Con A-stimulated guinea pig spleen cells. A high level of gpRANTES expression in Escherichia coli was achieved by mutation of a human RANTES (hRANTES) expression construct to obtain a 68-amino acid protein identical with the predicted guinea pig amino acid sequence, assuming an equivalent amino terminus as the human protein. Purified gpRANTES was an effective stimulus of human eosinophils as assessed by increases in intracellular free calcium in fura-2-loaded cells and chemotactic responses in vitro. gpRANTES exhibits similar potency and efficacy to hRANTES. In marked contrast, neither gpRANTES nor hRANTES was able to activate guinea pig peritoneal eosinophils in these assays, even in the presence of IL-5. However, gpRANTES was found to be a potent stimulator of guinea pig peritoneal macrophages. Following tracheal instillation of gpRANTES, a dose-dependent increase in macrophages, but not eosinophils, was observed in gpBAL. Macrophage accumulation was detectable by 6 h and sustained for at least 48 h. These results indicate that RANTES in the guinea pig may have a different cellular selectivity than that described in the human, which may be important in the use of animal models in the analysis of allergic disorders. These selectivities do not appear to be accounted for by differences in guinea pig and human RANTES sequences.
T cells bearing Vbeta8 are preferentially infected with exogenous mouse mammary tumor virus.
Authors: Upragarin N , Nishimura H , Wajjwalku W , Ando Y , Nagafuchi S , Watanabe T , Yoshikai Y
Published: December 2022

We previously reported that a mouse mammary tumor virus (MMTV(II-TES14)), encoding a superantigen specific for TCR Vbeta14, can infect lymph node (LN) cells of mice in an I-E-independent manner. Here we examined the kinetics of cell types infected with exogenous MMTV in the draining LN after s.c. injection of II-TES milk containing MMTV(II-TES14). The infectivity was assessed in LN cells sorted into each cell subset by a semiquantitative analysis of MMTV provirus using PCR with a primer specific for MMTV(II-TES14). Only B cells in the LN were infected by the MMTV on day 6 after injection, but CD8+ T cells and, to a lesser extent, CD4+ T cells were also found to be detectably infected on day 14 after the injection of II-TES milk. Among the T cells we examined, Vbeta8 T cells were most preferentially infected with MMTV, but no Vbeta14 T cells specific for MMTV(II-TES14) superantigen were infected on day 14 after infection. The transfer of Vbeta8 T cells sorted from mice injected with II-TES milk 14 days previously resulted in the deletion of CD4+ Vbeta14 T cells and in the MMTV infection of normal B6 mice. No MMTV infection of T cells occurred in IgM knockout mice, which lack a mature B cell compartment. These results suggest that MMTV surviving in B cells is transferred to Vbeta8 T cells, which may play an important role in MMTV longevity.
Antibodies against sialophorin (CD43) enhance the capacity of dendritic cells to cluster and activate T lymphocytes.
Authors: Fanales-Belasio E , Zambruno G , Cavani A , Girolomoni G
Published: December 2022

CD43 is a leukocyte membrane glycoprotein rich in negatively charged sialic acid residues that may act as an anti-adhesion molecule. In this study, we analyzed the expression and function of CD43 on epidermal Langerhans cells (LC) and dendritic cells derived from adherent peripheral blood monocytes treated with granulocyte-macrophage CSF and IL-4 (Mo-DC). Both freshly isolated LC (fLC) and Mo-DC expressed uniformly high amounts of membrane CD43, with levels unchanged in 48-h cultured LC or in TNF-alpha-treated Mo-DC. When fLC or Mo-DC were transiently preincubated with anti-CD43 mAb or neuraminidase, they formed much larger clusters with T cells as compared with cells incubated with control mAb. Moreover, a significantly increased proliferation of naive allogeneic T cells in a primary MLR assay was induced. Anti-CD43-treated Mo-DC also presented tetanus toxoid to primed T cells more efficiently. Immunofluorescence and immunogold electron microscopy showed that following Ab cross-linking and warming to 37 degrees C, CD43 is progressively removed from the cell surface and is internalized within endosomes and vacuoles. In addition, fLC and Mo-DC incubated with anti-CD43 mAb increased expression of HLA-DR, ICAM-1, B7-1, B7-2, CD40, and CD83 after 24 h, and this was partially prevented by culturing cells in the presence of cycloheximide. We thus postulate that membrane CD43, other than serving as anti-adhesion molecule, can mediate activation signals in immature DC, leading to augmented expression of adhesion/costimulatory molecules and Ag-presenting function.
Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice.
Authors: Pulendran B , Lingappa J , Kennedy M K , Smith J , Teepe M , Rudensky A , Maliszewski C R , Maraskovsky E
Published: December 2022

We have recently shown that Flt3 ligand administration dramatically increases dendritic cell (DC) numbers in various mouse tissues. This has enabled the identification of distinct mature DC subpopulations. These have been designated: population C (CD11c(bright) CD11b(bright)), D (CD11c(bright) CD11b(dull)), and E (CD11c(bright) CD11b(negative)) This report demonstrates that the mature DC subsets (C, D, and E) from Flt3 ligand-treated mice differ with respect to phenotype, geographic localization, and function. The myeloid Ags CD11b, F4/80, and Ly-6C are predominantly expressed by population C, but not D or E. In addition, a subset of population C-type DC expresses 33D1 and CD4. In contrast, DC within population D and E selectively express the lymphoid-related DC markers CD8alpha, DEC 205, CD1d, as well as CD23, elevated levels of CD117 (c-kit), CD24 (HSA), CD13, and CD54. Immunohistology indicates that the different DC subsets reside in distinct microenvironments, with populations D and E residing in the T cell areas of the white pulp, while DC within population C localize in the marginal zones. These DC subpopulations showed different capacities to phagocytose FITC-zymosan and to secrete IL-12 upon stimulation with Staphylococcus aureus cowan I strain + IFN-gamma + granulocyte-macrophage-CSF. Population C-type DC were more phagocytic but secreted little inducible IL-12 while population D- and E-type DC showed poor phagocytic capacity and secreted considerably higher levels of IL-12. These results underscore the importance of viewing DC development in vivo, as an interplay between distinct lineages and a maturational dependence on specific microenvironmental signals.
Regulation of tissue inhibitor of metalloproteinase-1 in fibroblasts and acute phase proteins in hepatocytes in vitro by mouse oncostatin M, cardiotrophin-1, and IL-6.
Authors: Richards C D , Kerr C , Tanaka M , Hara T , Miyajima A , Pennica D , Botelho F , Langdon C M
Published: December 2022

Mouse oncostatin M (mOSM) has been recently cloned; however, its full spectrum of biologic functions has not been defined. To assess its potential role in inflammation, we have tested the activity of mOSM in vitro in regulation of fibroblasts and hepatic cells. At concentrations of 10 and 20 ng/ml, mOSM stimulates tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in NIH-3T3 mouse embryonic fibroblasts, rat lung fibroblasts, and rat synovial fibroblasts, whereas mouse cardiotrophin-1 (mCT-1) or human OSM (hOSM) did not. Similarly, only mOSM was able to induce transcription of chloramphenicol acetyl-transferase (CAT) in NIH-3T3 cells transfected with a minimal TIMP-1 promoter/CAT construct. Mouse OSM had strong action inducing primary rat hepatocyte cultures to produce acute phase proteins; however, mOSM was very weak in its ability to stimulate acute phase protein synthesis in rat H35 cells or human HepG2 cells, which was consistent with weak STAT activation in H35 cells and HepG2 cells. Binding studies showed that NIH-3T3 cells possessed high affinity binding sites for mOSM, but rat H35 cells did not. On the other hand, mCT-1 and mouse IL-6 induced strong STAT activation as well as marked increases in acute phase protein production by H35 cells. These results indicate that mOSM does not share a functional receptor with mCT-1 or hOSM in mouse and rat cells and that hOSM does not activate the putatively specific OSM receptor on mouse or rat cells. These results also suggest that mOSM is an important cytokine in inflammation, through modulation of fibroblast function as well as hepatocyte responses.
Changes in cytokine secretion induced by altered peptide ligands of myelin basic protein peptide 85-99.
Authors: Ausubel L J , Krieger J I , Hafler D A
Published: December 2022

Myelin basic protein (MBP)-specific T cells were isolated directly from peripheral blood by stimulation either with native MBPp85-99 or altered peptide ligands (APLs) in which substitutions of the lysine were made at position 93, a TCR contact residue. We report here that the APL 93A could alter the cytokine profile of some autoreactive MBPp85-99 reactive T cell clones, switching them from a Th0 phenotype secreting high concentrations of IL-4, IL-5, and IFN-gamma into Th2 cells secreting significantly less IFN-gamma. However, in vitro stimulation with the 93A peptide, in some instances, induced T cells that responded better to the native MBPp85-99 peptide. Functionally, the APL 93A was shown to act as an antagonist for IFN-gamma secretion. Based on TCR sequencing and single cell cloning, this alteration in cytokine profile was determined to be the result of the differential activation of individual T cell clones. We discuss the implications of these data for the strength of signal model of T cell activation.
Isolation and characterization of Exodus-2, a novel C-C chemokine with a unique 37-amino acid carboxyl-terminal extension.
Authors: Hromas R , Kim C H , Klemsz M , Krathwohl M , Fife K , Cooper S , Schnizlein-Bick C , Broxmeyer H E
Published: December 2022

Chemokines are a group of small, homologous proteins that regulate leukocyte migration, hemopoiesis, and HIV-1 absorption. We report here the cloning and characterization of a novel murine and human C-C chemokine termed Exodus-2 for its similarity to Exodus-1/MIP-3alpha/LARC, and its chemotactic ability. This novel chemokine has a unique 36 or 37 (murine and human, respectively) amino acid carboxyl-terminal extension not seen in any other chemokine family member. Purified recombinant Exodus-2 was found to have two activities classically associated with chemokines: inhibiting hemopoiesis and stimulating chemotaxis. However, Exodus-2 also had unusual characteristics for C-C chemokines. It selectively stimulated the chemotaxis of T-lymphocytes and was preferentially expressed in lymph node tissue. The combination of these characteristics may be a functional correlate for the unique carboxyl-terminal structure of Exodus-2.
Low affinity of cell surface lymphocyte function-associated antigen-1 (LFA-1) generates selectivity for cell-cell interactions.
Authors: Ganpule G , Knorr R , Miller J M , Carron C P , Dustin M L
Published: December 2022

We examined binding of soluble intercellular adhesion molecule-1 (ICAM-1) dimers and a range of ICAM-1-coated particles to activated T cells. Lymphocyte function-associated antigen-1 (LFA-1) on the surface of activated T cells did not bind soluble ICAM-1 dimers with high affinity. In contrast, activated T cells adhered avidly to ICAM-1-coated planar surfaces. Between these two extremes, a range of ICAM-1-bearing particles was tested for binding. Activated T cells bound particles of 1-microm diameter or larger, but did not bind particles of 0.5-microm diameter or smaller. This threshold was eliminated, and all forms of ICAM-1 bound to LFA-1 when LFA-1 was converted to a high affinity form with an activating antibody. We show that high affinity LFA-1 is generated only as a consequence of an initial low affinity interaction of LFA-1 with ICAM-1 under physiological conditions. Therefore, the selectivity of cell surface LFA-1 for cell-sized particles bearing ICAM-1 appears to be due to the maintenance of low affinity LFA-1 on the surface of activated T cells. These findings alter the definition of inside-out signaling for LFA-1.
Early neutralizing antibody response against mouse mammary tumor virus: critical role of viral infection and superantigen-reactive T cells.
Authors: Luther S A , Maillard I , Luthi F , Scarpellino L , Diggelmann H , Acha-Orbea H
Published: December 2022

Infectious mouse mammary tumor virus (MMTV) is a retrovirus that expresses a superantigen shortly after infection of B cells. The superantigen first drives the polyclonal activation and proliferation of superantigen-reactive CD4+ T cells, which then induce the infected B cells to proliferate and differentiate. Part of the MMTV-induced B cell response leads to the production of Abs that are specific for the viral envelope protein gp52. Here we show that this Ab response has virus-neutralizing activity and confers protection against superinfection by other MMTV strains in vivo as soon as 4 to 7 days after infection. A protective Ab titer is maintained lifelong. Viral infection as well as the superantigen-induced T-B collaboration are required to generate this rapid and long lasting neutralizing Ab response. Polyclonal or superantigen-independent B cell activation, on the contrary, does not lead to detectable virus neutralization. The early onset of this superantigen-dependent neutralizing response suggests that viral envelope-specific B cells are selectively recruited to form part of the extrafollicular B cell response and are subsequently amplified and maintained by superantigen-reactive Th cells.
Heterogeneity of biologic responses of melanoma-specific CTL.
Authors: Fonteneau J F , Le Dréan E , Le Guiner S , Gervois N , Diez E , Jotereau F
Published: December 2022

To better understand how Ag density influences the various biologic responses of CTL, we analyzed lysis and, at the single cell level, cytokine production by a panel of melanoma-specific CTL clones. Titration experiments done with peptide-pulsed TAP-deficient T2 cells indicated that: 1) Ag density affects both the fraction of responding cells and the amount of cytokine secreted by each cell. 2) Different responses have a relative Ag requirement that may vary between clones. Lysis and secretion of IFN-gamma, and for most clones' secretion of TNF-alpha, required lower Ag densities, by one or two logs, than IL-2 and granulocyte-macrophage CSF secretion. 3) In a significant fraction of IFN-gamma-secreting cells, IL-2 production is not induced. 4) A large fraction of cloned cells is refractory to lymphokine gene activation for about 2 wk after previous stimulation. Together these data indicate that CTL biologic responses are controlled by variable Ag thresholds and by additional parameters affecting activation requirements of each cell. A similar heterogeneity of cytokine responses was observed to Ag endogenously presented by melanoma cells. As a consequence, most melanoma lines, including those with the highest Ag expression, could trigger only low CTL fractions to secrete IL-2 and, also for most clones, granulocyte-macrophage CSF. This may be an important component of the inefficiency of specific CTL in cancer patients.