bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of sialoadhesin expression on rat macrophages. Induction by glucocorticoids and enhancement by IFN-beta, IFN-gamma, IL-4, and lipopolysaccharide.
Authors: van den Berg T K , van Die I , de Lavalette C R , Döpp E A , Smit L D , van der Meide P H , Tilders F J , Crocker P R , Dijkstra C D
Published: December 2022

Sialoadhesin is a macrophage-restricted member of the Ig superfamily that mediates adhesion with lymphoid and myeloid cells. It is expressed on a subpopulation of macrophages in lymphoid tissues and in chronic inflammation (e.g., during autoimmune diseases). We have studied the regulation of sialoadhesin expression in vitro and show that glucocorticoids (GC) induce sialoadhesin expression on freshly isolated rat macrophages and the rat macrophage cell line R2. The cytokines IFN-beta, IFN-gamma, IL-4, and LPS, although unable to induce sialoadhesin expression by themselves, were able to enhance GC-mediated induction of sialoadhesin. Sialoadhesin expression was functional as shown by cell adhesion assays with human RBCs. Northern blotting experiments indicated that regulation predominantly occurred at the mRNA level. Comparison of the different combinations of GC and cytokines/LPS revealed differences in the level of GC-dependent enhancement of sialoadhesin expression, with IFN-beta and IL-4 being more potent than IFN-gamma and LPS. Moreover, the effects of IFN-gamma and LPS could be reproduced by priming, whereas IFN-beta and IL-4 were required simultaneously with GC. The regulation of sialoadhesin expression was mediated by the GC receptor, and not by mineralocorticoid receptor, as shown by inhibition experiments with specific antagonists. Finally, it is demonstrated that macrophages in the adrenal gland, the major site of endogenous GC production, express sialoadhesin. This study demonstrates that GC act as a primary inducer of sialoadhesin expression on rat macrophages, and that the response can be enhanced by IFN-beta, T cell-derived cytokines, or LPS.
Molecular analysis of stimulatory anti-thyrotropin receptor antibodies (TSAbs) involved in Graves' disease. Isolation and reconstruction of antibody genes, and production of monoclonal TSAbs.
Authors: Akamizu T , Matsuda F , Okuda J , Li H , Kanda H , Watanabe T , Honjo T , Mori T
Published: December 2022

Anti-thyrotropin (TSH) receptor autoantibodies (TRAbs) have been known to be involved in Graves' disease. To understand the molecular mechanism for pathogenesis of TSAbs in Graves' disease, we isolated and reconstituted the Ig genes of EBV-transformed B cell clones producing monoclonal thyroid stimulating Ab (TSAb) obtained from patients with Graves' disease. The V region genes of Ig heavy (H) and light (L) chains of two TSAb clones, IgG clone B6B7 and IgM clone 101-2, were isolated by the PCR. Nucleotide sequencing analysis revealed that germ-line VH and VK segments widely used for autoantibodies including the previously isolated TRAbs were utilized in the two clones. A significant number of somatic mutations were found in V regions of both clones, indicating the involvement of somatic mutations for the TSAb specificity. Reconstituted Ig H and L chain genes of the two clones were stably introduced into myeloma cells for IgG1 production. IgGs purified from cultured supernatants of both transfectants exhibited significant TSAb activities, while they did not inhibit TSH binding to the receptor. The successful expression of recombinant TSAbs in eukaryotic cells will provide opportunities to apply them to various pathophysiologic, diagnostic and therapeutic investigations in autoimmune thyroid diseases.
Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response.
Authors: Early G S , Zhao W , Burns C M
Published: December 2022

Systemic lupus erythematosus is characterized by B cell production of pathogenic autoantibodies dependent upon cooperation from CD4+ Th cells. The interaction between CD40 on B cells and CD40 ligand (CD40L) on Th cells is necessary for normal thymus-dependent Ab production. An anti-murine CD40L mAb blocks binding of CD40L to CD40 and prevents primary and secondary immune responses to thymus-dependent Ags. In this study, New Zealand Black x New Zealand White lupus-prone mice treated with this anti-CD40L Ab from ages 4 to 10 mo had reduced anti-DNA autoantibody production and renal disease and significantly prolonged survival compared with control mice. Pathologic examination verified the absence of significant renal damage or immune deposition in responding mice. Mice that responded to treatment did not develop an Ab response to the administered Ab. Long-term survivors mounted a substantial Ab response to keyhole limpet hemocyanin after completion of anti-CD40L Ab treatment, suggesting that some of the immunosuppressive effects of the Ab may be reversible. These results suggest a human form of this Ab may have therapeutic utility in human systemic lupus erythematosus.
Attenuation of collagen-induced arthritis in 55-kDa TNF receptor type 1 (TNFR1)-IgG1-treated and TNFR1-deficient mice.
Authors: Mori L , Iselin S , De Libero G , Lesslauer W
Published: December 2022

The role of TNF and its type 1 receptor (TNFR1) in the pathogenesis of collagen-induced arthritis (CIA) was investigated in mice using two approaches. First, DBA/1 mice were treated after immunization with type II collagen by injecting TNFR1-IgG1 fusion protein to neutralize systemic TNF. CIA was prevented when treatment was administered shortly before the onset of clinical disease, suggesting that TNF is a crucial mediator in the late initiation phase of the arthritic process. In a second approach, TNFR1-deficient mice, generated by gene targeting and crossed to DBA/1, were used. These mice developed CIA with a low incidence and in a milder form. However, once a joint was afflicted, the disease progressed in this joint to the same end stage as that in wild-type mice. These data suggest that TNFR1 is the main transducer of TNF proinflammatory effects establishing CIA, but the progression of arthritis to tissue destruction and ankylosis is independent of TNFR1.
Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity.
Authors: Fang N , Motto D G , Ross S E , Koretzky G A
Published: December 2022

SLP-76 (SH2 domain leukocyte protein of 76 kDa) is a recently identified substrate of the TCR-stimulated protein tyrosine kinases that functions in the signal transduction cascade linking the TCR with IL-2 gene expression. In this report, we demonstrate that engagement of the TCR results in tyrosine phosphorylation of SLP-76 in its amino-terminal acidic region. Two tyrosines (Y113 and Y128) fall within an identical five amino-acid motif and are shown to be phosphorylated upon TCR ligation. Although mutation of either Y113 and Y128 has a minimal effect on SLP-76 function, mutation of both residues decreases significantly the ability of SLP-76 to promote T cell activation. A third tyrosine within the amino-terminal region (Y145) appears to be the most important for optimal SLP-76 function, as altering it alone to phenylalanine has a potent impact on SLP-76 augmentation of NFAT promoter activity.
Mechanisms of tolerance induction: splenocytes from total lymphoid irradiated mice inhibit the IL-2 pathway in TCR-activated CD4 cells.
Authors: Field E H , Ye Z X , Rouse T M
Published: December 2022

Mice given total lymphoid irradiation (TLI) can be tolerized by an incompletely understood mechanism to foreign Ags given during a brief window of time immediately after completing radiation. We previously showed that splenocytes taken from mice immediately after completing TLI treatment (TLI cells) block the ability of control cells to proliferate or to produce IL-2 in response to alloantigen, suggesting that TLI cells interfere with the IL-2 pathway in responder lymphocytes. To further characterize the mechanism by which TLI cells affect IL-2, we utilized an in vitro system of T cell activation. Adding TLI cells into cultures decreased the ability of TCR-stimulated T cells to proliferate and to secrete IL-2 by 40 to 60%. Exogenous IL-2 did not restore the proliferative response. The inhibition of IL-2 secretion resulted both from a decrease in the number of IL-2-producing cells and a decrease in IL-2 mRNA transcription. TLI cells directly inhibited IL-2 secretion by TCR-stimulated CD4 cells, as determined by ELISPOT. Competitive PCR demonstrated that TLI cells decreased IL-2 transcription of TCR-stimulated CD4 cells by 75%. In situ hybridization confirmed that unfractionated spleen and sorted CD4 cells from TCR-stimulated cultures transcribed less IL-2 RNA in the presence of TLI cells. The results show that TLI cells directly inhibit IL-2 transcription and protein secretion of normal TCR-stimulated CD4 cells.
Porcine endothelial CD86 is a major costimulator of xenogeneic human T cells: cloning, sequencing, and functional expression in human endothelial cells.
Authors: Maher S E , Karmann K , Min W , Hughes C C , Pober J S , Bothwell A L
Published: December 2022

Porcine aortic endothelial cells (PAECs), unlike human endothelial cells, express a surface protein recognized by human CTLA4Ig fusion protein that costimulates human T cells through CD28. We have cloned porcine CD86 (pCD86) from an immortalized porcine endothelial cell line, PEC-A, that expresses high levels of this CTLA-4-binding protein. pCD86 mRNA is expressed in PEC-A and PAECs but not in human endothelial cells. Expression of stably transfected pCD86 in CHO cells modestly costimulates human T cell proliferation and IL-2 secretion. Expression of transiently transfected pCD86 in human umbilical vein endothelial cells strongly costimulates IL-2 production by human T cells, comparable to costimulation by PAECs. Costimulation of human T cells by pCD86 in both systems is as effective as costimulation by human CD80 or CD86, and can be blocked by human CTLA4Ig. We conclude that pCD86 contributes to the strong xenoreactivity of porcine endothelium.
Toxic shock syndrome toxin-1-induced death is prevented by CTLA4Ig.
Authors: Saha B , Jaklic B , Harlan D M , Gray G S , June C H , Abe R
Published: December 2022

Lethal toxic shock syndrome (TSS) results from the MHC class II presentation of bacterial superantigens, most commonly toxic shock syndrome-1 (TSST-1), to specific TCR Vbeta-bearing T cells. This superantigen-induced stimulation of whole T cell subsets leads to the exuberant cytokine production that in turn causes the shock syndrome. Since T cell activation and cytokine production are known to be dependent upon costimulatory signals, we reasoned that interfering with costimulation could effect TSS outcome. To test that hypothesis, we evaluated the effect of CTLA4Ig, a fusion protein known to block costimulatory signaling, on TSST-1-induced responses. CTLA4Ig not only blocked TSST-1-stimulated T cell proliferation by 90% in vitro, it also strikingly ameliorated TSST-1 induced TSS in vivo. While all mice co-administered TSST-1 and control Ig died, 75% of the CTLA4Ig plus TSST-1-treated mice survived. This salutary CTLA4Ig effect correlated with markedly diminished TSST-1 induced serum levels of TNF-alpha and IFN-gamma, but TSST-1-triggered IL-2 release was not affected. Surprisingly, while CTLA4Ig treatment group survivors remained sensitive to TSS induced by an unrelated superantigen (staphylococcal enterotoxin B), they were completely resistant to a second TSST-1 challenge. Furthermore, this TSST-1 resistance could be transferred to naive C57BL/6 mice using CD8+ T cells from CTLA4Ig plus TSST-1-primed mice. These data suggest several novel interpretations: 1) that the release of TNF-alpha and IL-2 have a different costimulatory signal dependence in vivo, 2) that the TSS resistance conferred by CTLA4Ig was superantigen specific, and 3) that the delayed and transferable resistance to TSST-1 was due, at least in part, to CD8+ T cells with suppressor function.
MHC genotype controls the capacity of ligand density to switch T helper (Th)-1/Th-2 priming in vivo.
Authors: Schountz T , Kasselman J P , Martinson F A , Brown L , Murray J S
Published: December 2022

A quantitative mechanism for the differentiation of CD4 T cells into recognized subsets of Th1 and Th2 effectors is controversial. Here, we define the Ag dose more precisely to the density of a minimal immunogenic peptide presented on the surface of a specific APC type. Th1 and Th2 responder MHC genotypes differ by as much as an order of magnitude in the density of this peptide displayed on B7-2+ B cells. We asked whether such B cells presenting a low ligand density primed Th2 effectors in an MHC genotype with predisposed high-density presentation and Th1-type immunity, and whether high ligand density B cells primed Th1 effectors in an MHC genotype that normally presents a low density and the Th2 phenotype. While low ligand density had the capacity to switch phenotype in the Th1 responder, high-density presentation did not alter genetically determined Th2 responder status.
Apoptosis of mouse naive T cells induced by recombinant soluble Fas ligand and activation-induced resistance to Fas ligand.
Authors: Suda T , Tanaka M , Miwa K , Nagata S
Published: December 2022

We have investigated the susceptibility of mouse T cells at various maturation and activation stages to recombinant soluble mouse Fas ligand (termed WX1). CD4+CD8+ thymocytes were preferentially killed by WX1, although CD4+CD8- and CD4-CD8+ thymocytes were also killed at higher concentrations of WX1. The majority of CD4-CD8- thymocytes were resistant to the same doses of WX1. Naive peripheral T cells were also killed by WX1 at concentrations similar to those for single-positive thymocytes. Activation-induced cell death of previously activated T cells is known to be mediated by Fas and Fas ligand. However, the activation of naive splenic T cells by mAbs against CD3/TCR complex rather induced resistance against WX1. This resistance was induced in activated T cells but not in bystander T cells. These mechanisms seem to explain why activated T cells are not immediately killed and why bystander T cells are not activated in an Ag-nonspecific manner.
Cloning of minimally divergent allelic forms of the natural killer (NK) receptor Ly-49C, differentially controlled by host genes in the MHC and NK gene complexes.
Authors: Sundbäck J , Kärre K , Sentman C L
Published: December 2022

We have investigated whether expression of Ly-49C, detected by the mAb SW5E6, is subject to similar regulation by host MHC as is Ly-49A. Ly-49C expression was regulated by host MHC differentially in mice of A/Sn (A) and C57BL/6 (B) origin. Analysis of AXB recombinant inbred strains suggested two loci regulating the expression, one segregating with the NK gene complex and the other with the MHC. In MHC congenic strains, the A form of Ly-49C had a low expression level and was further down-regulated in the presence of the H-2b haplotype (very low). The B form of Ly-49C had a high expression level and was down-regulated in the presence of H-2Kk (medium). To test the hypothesis that the Ly-49C receptor in A/Sn and C57BL/6 mice may be differentially regulated by MHC products because they represent allelic forms, cDNAs for Ly-49C were cloned by reverse-transcriptase PCR from A/Sn and C57BL/6 mouse strains. The Ly-49C sequences from the two strains differed by six nucleotides leading to four predicted amino acid changes. Both cDNAs could be detected by the Ly-49C-specific Ab when expressed in EL-4 cells. Thus, like Ly-49A, Ly-49C expression is regulated by MHC class I molecules. Furthermore, A/Sn and C57BL/6 mice express different forms of Ly-49C that are down-regulated by different MHC class I molecules.
Lack of requirement for SHP-1 in both Fas-mediated and perforin-mediated cell death induced by CTL.
Authors: Takayama H , Lee M H , Shirota-Someya Y
Published: December 2022

The Fas (CD95)-transmitted cell death signal has been reported to involve a protein tyrosine phosphatase, SHP-1. We analyzed the role of SHP-1 in the Fas-dependent as well as the perforin-dependent pathways of CTL-mediated killing using target cells prepared from SHP-1-deficient motheaten mice. Con A blast targets prepared from both a motheaten mouse and a phenotype-normal littermate were equally sensitive to the cytolysis and DNA fragmentation induced by both perforin-deficient Fas-dependent CTL and Fas ligand-deficient perforin-positive CTL. Fas-induced DNA degradation detected by the terminal deoxynucleotide transferase reaction was also observed in the killing of motheaten thymocytes by a Fas-based CTL as well as by anti-Fas mAb. These data cast doubt on the involvement of SHP-1 in Fas-induced lymphoid cell death.
Mutations in the MHC class II binding domains of staphylococcal enterotoxin A differentially affect T cell receptor Vbeta specificity.
Authors: Newton D W , Dohlsten M , Olsson C , Segrén S , Lundin K E , Lando P A , Kalland T , Kotb M
Published: December 2022

C-terminal residues of staphylococcal enterotoxin A (SEA), including H187, D225, and D227, are involved in moderate affinity binding to MHC class II beta-chain, whereas N-terminal residues, including F47, are involved in low affinity binding to MHC class II alpha-chain. The effect of alanine substitutions at residues D227 or F47 on induction of T cell proliferation and the expansion of specific TCR Vbeta families was determined. SEA wild type specifically activated T cells expressing Vbeta1, Vbeta5.2, Vbeta6, Vbeta7, Vbeta9, Vbeta18, and Vbeta22. Although SEA-D227A exhibited substantially reduced mitogenicity compared with SEA wild type, it expanded the same Vbeta-bearing T cells, except those expressing Vbeta1. By contrast, SEA-F47A, which was slightly less mitogenic than SEA wild type, induced expansion only of T cells expressing Vbeta6, Vbeta7, and to a lesser extent Vbeta22. Therefore, specific mutations affecting either MHC class II alpha or beta binding sites differentially affect the Vbeta specificity of this superantigen. The lack of expansion in four of seven Vbeta families by SEA-F47A suggests that the class II alpha binding site may position SEA on the MHC class II molecules in an appropriate conformation for interaction with certain Vbeta elements.
The progeny of a single virgin B cell predominates the human recall B cell response to the capsular polysaccharide of Haemophilus influenzae type b.
Authors: Barington T , Hougs L , Juul L , Madsen H O , Ryder L P , Heilmann C , Svejgaard A
Published: December 2022

Restricted V region diversity is a key feature of Abs to many haptens and simple polysaccharides. Two possible mechanisms exist: 1) selection of many clonally unrelated B cells using very similar or identical VDJ and VJ rearrangements; and 2) selection of a heavily expanded progeny of few virgin B cells. How many virgin B cells eventually give rise to the total Ab response to a simple Ag is a fundamental immunologic question. In this report, we address this question in human adults by analyzing the rearranged VkappaJkappa genes of B cells responding to a single dose of the capsular polysaccharide of Haemophilus influenzae type b coupled to tetanus toxoid. We combined affinity purification of circulating vaccine-induced Ab-secreting cells with PCR amplification of cDNA followed by cloning and sequencing. Forty-eight and 42 kappa VJ gene transcripts were analyzed from two adults, respectively. Both individuals used extremely restricted repertoires with >90% of the cells using a single Vkappa gene rearranged to a single Jkappa gene. Despite the fact that the Ab responses engaged high numbers of Ab-secreting cells, analysis of the many shared, somatically acquired mutations showed that the majority of the cells originated from a common virgin B cell. Kinetic considerations implied that an extremely selected population of hypermutated memory B cells must have existed in these individuals before the first systemic immunization with the Ag. A possible role for the mucosal immune system in the priming and selection of these cells is proposed.
Cytokine expression by human peripheral blood dendritic cells stimulated in vitro with HIV-1 and herpes simplex virus.
Authors: Ghanekar S , Zheng L , Logar A , Navratil J , Borowski L , Gupta P , Rinaldo C
Published: December 2022

Dendritic cells are potent stimulators of Ag-specific T cell responses and have been implicated in the pathogenesis of HIV-1 and other viral infections. Although cytokines may be involved in both of these processes, there is little information on the expression of cytokines by human blood dendritic cells. We characterized cytokine gene and protein expression in dendritic cells that were purified from normal human PBMC by flow cytometry and stimulated in vitro for up to 24 h with HIV-1 or herpes simplex virus (HSV). The unstimulated, uncultured dendritic cells were defined by their phenotype (HLA DR+ CD3- CD19- CD16- CD56- CD14-) and distinct morphology, lack of mRNA expression for CD3, CD14 and CD19, and presence of mRNA for CD4 and CD83. The purified dendritic cells also expressed CD4 (70-90%), CD33 (36-48%), and CD11c (44-54%); lacked CD1a expression (<1%); and were potent stimulators of an allogeneic MLR. The stimulated dendritic cells expressed mRNA for IFN-alpha, IL-1alpha, IL-1beta, IL-6, IL-10, IL-12, GM-CSF, and TNF-alpha within 4 to 12 h as determined by reverse transcription-PCR, with higher levels induced by HSV compared with HIV-1 strains IIIb or BaL. In contrast, the dendritic cells produced detectable protein only for IFN-alpha and IL-6 in response to HIV-1 or HSV, and IL-1beta in response to HSV within 24 h. There were no differences in expression of CD80 and CD86 surface molecules by dendritic cells that were either mock stimulated or stimulated with HIV-1 or HSV for 24 h. Production of IFN-alpha, IL-1beta, and IL-6 by dendritic cells may be important to the immunologic function of these cells and their role in the pathogenesis of HIV-1 and HSV infections.
The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs.
Authors: Binns R M , Whyte A , Licence S T , Harrison A A , Tsang Y T , Haskard D O , Robinson M K
Published: December 2022

We have studied the role of E-selectin in leukocyte accumulation into Ag-specific cutaneous delayed-type hypersensitivity reactions in pigs sensitized to the topical application of 2,4-dinitro-1-fluorobenzene or to the intradermal injection of bacillus Calmette-Guérin. The delayed-type hypersensitivity reactions were shown to be specific for the sensitizing Ag and characterized by the up-regulation of E-selectin, as demonstrated by the uptake of tracer 99mTc-labeled monoclonal anti-E-selectin mAb and entry of 51Cr-labeled PBL and (111)In-labeled polymorphonuclear cells (PMN). Intravenous injection of 5 mg/kg of a F(ab')2 preparation of a monoclonal anti-E-selectin Ab at peak times of leukocyte entry resulted in a significant inhibition of entry of both PMN and lymphocytes. The anti-E-selectin Ab inhibited PMN recruitment by 70 to 90% and lymphocyte recruitment by 50 to 60%. In comparison, an anti-CD18 treatment reduced PMN recruitment by 70 to 90% and lymphocyte recruitment by 60 to 70% in this model. These data confirm an important role for E-selectin in the recruitment of both PMN and lymphocytes to sites of immune-based dermal inflammation.
Monocytes can phagocytose Gram-negative bacteria by a CD14-dependent mechanism.
Authors: Grunwald U , Fan X , Jack R S , Workalemahu G , Kallies A , Stelter F , Schütt C
Published: December 2022

Phagocytosis of bacteria by monocytes and neutrophil granulocytes provides an important first line of defense against bacterial infections. Opsonization of bacteria with complement and phagocytosis by neutrophils is dependent on divalent cations and does not take place in blood that has been anticoagulated with EDTA. Monocytes, however, do carry out phagocytosis even in the presence of EDTA. We show here that this divalent cation-independent phagocytosis pathway requires the presence of the LPS receptor CD14 on the cell surface. This pathway is dependent on the availability of LPS binding protein, can be blocked by anti-CD14 Abs, by an excess of soluble CD14, by excess free LPS, or by an excess of unlabeled Gram-negative bacteria. In contrast, intact Gram-positive bacteria fail to inhibit this process. These experiments define a CD14-dependent phagocytosis pathway for Gram-negative bacteria that operates in monocytes in human whole blood. This pathway may be able to deal with bacterial pathogens that have developed resistance to complement-dependent opsonization and phagocytosis by neutrophils.
Human RANTES acts as a receptor antagonist for guinea pig eotaxin in vitro and in vivo.
Authors: Marleau S , Griffiths-Johnson D A , Collins P D , Bakhle Y S , Williams T J , Jose P J
Published: December 2022

The guinea pig C-C chemokine, eotaxin, is a potent and selective eosinophil chemoattractant in guinea pig airways and skin in vivo, and stimulates both guinea pig and human eosinophils in vitro. The human C-C chemokine RANTES (30% homology with guinea pig eotaxin) stimulates human eosinophils in vitro, but does not stimulate guinea pig eosinophils, even though these cells bind 125I-RANTES. Similar concentrations of eotaxin and unlabeled RANTES competitively inhibit the binding of 125I-RANTES to guinea pig eosinophils, suggesting that eotaxin and RANTES share a common binding site on these cells. In the present study, we investigated the possibility that human RANTES, binding to a putative eotaxin receptor on guinea pig eosinophils, might block functional responses to eotaxin. When fura-2-loaded cells were first exposed to RANTES, which failed to elevate the intracellular calcium concentration, the response to a subsequent challenge with eotaxin was inhibited in a dose-dependent manner. Inhibition was also demonstrated when the two chemokines were added simultaneously. Another human C-C chemokine, MCP-3 (52% homology with guinea pig eotaxin), had similar inhibitory effects on the eotaxin-induced activation of guinea pig eosinophils in vitro. RANTES inhibited (111)In-eosinophil accumulation in response to intradermal eotaxin in vivo. In contrast, RANTES had no significant effect on responses to leukotriene B4 in vitro or in vivo. Thus, these experiments in the guinea pig demonstrate that human RANTES is the first prototypic antagonist of an eotaxin receptor.
Immunomodulatory effects of IL-12 on allergic lung inflammation depend on timing of doses.
Authors: Sur S , Lam J , Bouchard P , Sigounas A , Holbert D , Metzger W J
Published: December 2022

We investigated the effects of IL-12 on a murine model of allergic lung inflammation. Administration of IL-12 was timed to interfere with either allergic sensitization (early dosage) or the hypersensitivity inflammatory response in the lung (late dosage), or both (early and late dosages). Comparisons of IL-12- and PBS-treated animals within each treatment group revealed several noticeable effects of IL-12. Early dosage, and the combination of early and late dosages, strikingly decreased ragweed-specific serum IgE, tracheal ring reactivity to acetylcholine, and BAL eosinophilia following allergen challenge. In contrast, late dosage had no effect on IgE levels and only a minimal effect on tracheal ring reactivity, but had a modest effect on recruitment of eosinophils. Early dosage down-regulated IL-5 and IL-10, but did not alter IL-4 or IFN-gamma expression. Late dosage down-regulated IL-5, up-regulated IL-10 and IFN-gamma, but did not change IL-4 expression. The combination of early and late dosage down-regulated IL-4, IL-5, and IL-10 expression, but increased IFN-gamma expression and production in the BAL cells and fluids. Taken together, these results indicate that IL-12 has potent immunomodulatory effects on allergic lung inflammation that depend on the timing of IL-12 administration relative to allergic sensitization and allergen challenge.
Disease-specific changes in gammadelta T cell repertoire and function in patients with pulmonary tuberculosis.
Authors: Li B , Rossman M D , Imir T , Oner-Eyuboglu A F , Lee C W , Biancaniello R , Carding S R
Published: December 2022

Although gammadelta T cells are known to contain the highest frequency of mycobacteria-reactive cells in humans and numerous studies have suggested that they play an important role in the initial immune response to Mycobacterium tuberculosis (Mtb), very few studies have attempted to analyze these cells in patients with active pulmonary tuberculosis. The aim of the present study was therefore to evaluate the consequences of infection on the number and activity of mycobacteria-reactive gammadelta T cells. Three-color flow cytometric analysis of blood and bronchoalveolar lavage gammadelta T cells of patients diagnosed with active pulmonary tuberculosis showed that compared with normal healthy subjects and patients with the unrelated pulmonary granulomatous diseases sarcoidosis and berylliosis the size of the mycobacteria-reactive Vgamma9+/Vdelta2+ gammadelta T cell subset in both the blood and lung was dramatically reduced. In addition, the Vgamma9+/Vdelta2+ cells left intact in patients with tuberculosis were refractory to in vitro stimulation by Mtb Ags, which are potent stimuli for these cells in normal subjects. Our results demonstrate for the first time a strong correlation between the absence or loss of the major Vgamma9+/Vdelta2+ Mtb-reactive subset of gammadelta T cells and manifestations of disease, consistent with the hypothesis that these gammadelta T cells play a role in the protective immune response to Mtb infection.
A natural IgA-anti-F(ab')2gamma autoantibody occurring in healthy individuals and kidney graft recipients recognizes an IgG1 hinge region epitope.
Authors: Terness P , Navolan D , Moroder L , Siedler F , Weyher E , Kohl I , Dufter C , Welschof M , Drugarin D , Schneider F , Opelz G
Published: December 2022

Natural anti-IgG autoantibodies are found both in healthy individuals and in patients with certain diseases. One group of these Abs recognizes epitopes located in the F(ab')2 region of the IgG molecule. The immunoregulatory role of these Abs in healthy individuals, graft rejection, and disease was previously studied, usually with a focus on the characterization of anti-idiotypic Abs. In the present study, we characterize the epitope recognized by an anti-F(ab')2gamma autoantibody of the IgA isotype, which occurs in the serum of healthy individuals and kidney transplant recipients. The autoantibody described herein reacts strongly with F(ab')2gamma but only poorly with Fab(gamma) fragments, a binding pattern pointing to an epitope located in the hinge region. Using synthetic peptides, we identified a conformational epitope that overlaps the middle and part of the lower hinge region. Structural analyses of peptide constructs showed that a defined conformation of the first three residues of the lower hinge is required for a full expression of the epitope. Binding of IgA to the hinge region of IgG1 covers part of the physiologically active Fc domain, immobilizes the Fab arms, and thereby can be expected to exert immunoregulatory functions.
Differential effect of CTLA4Ig on murine graft-versus-host disease (GVHD) development: CTLA4Ig prevents both acute and chronic GVHD development but reverses only chronic GVHD.
Authors: Via C S , Rus V , Nguyen P , Linsley P , Gause W C
Published: December 2022

The role of costimulation was examined in an in vivo model of alloantigen-driven Th1 or Th2 cytokine responses, the parent-into-F1 model of acute or chronic graft-vs-host disease (GVHD), respectively. The soluble fusion protein, murine CTLA4Ig, which blocks engagement of CD28 by its natural ligand B7-1 and B7-2, was administered either early, at the time of GVHD induction, or delayed, after the establishment of Th1 or Th2 effector responses (day 7). Early administration of CTLA4Ig prevented the development of both acute and chronic GVHD by preventing the activation of donor T cells, i.e., by blocking characteristic Th1 or Th2 cytokine production and blocking memory marker up-regulation on donor T cells. Delayed CTLA4Ig administration was unable to alter acute GVHD but did reverse chronic GVHD as evidenced by normalization of serum autoantibody levels, normal host B cell numbers and MHC class II expression, reduced donor T cell expression of CD40 ligand, and reduced numbers of donor CD4+ memory T cells. The percentage of donor memory cells was not altered by delayed CTLA4Ig. We conclude that in this model, alloantigen-driven Th1 or Th2 responses are equally susceptible to costimulatory blockade at the onset of disease; however, once effector mechanisms become established, only Th2-driven responses have a requirement for further costimulation for the continued expansion of CD4+ T cells. These data suggest that humoral, lupus-like autoimmunity requires continuous T cell help for B cells, and agents that interrupt this process may be beneficial.
Dysregulated intrathymic development in the IL-2-deficient mouse leads to colitis-inducing thymocytes.
Authors: Lúdvíksson B R , Gray B , Strober W , Ehrhardt R O
Published: December 2022

Gene-targeted mice lacking the IL-2 gene (IL-2 -/- mice) develop various forms of autoimmunity as well as severe colitis, either spontaneously in a conventional environment or after immunization with 2,4,6-trinitrophenol (TNP)-conjugated keyhole limpet hemocyanin (KLH) in a specific pathogen-free environment. We show here that the induction of colitis with TNP-KLH induces a change in the thymocyte population characterized by decreased numbers of double positive (DP; CD4+CD8+) thymocytes (IL-2 +/+, 45.2 x 10(6) vs IL-2 -/-, 23.6 x 10(6)) and increased numbers of single positive (SP; CD4+CD8- or CD4-CD8+) thymocytes (IL-2 +/+, 5.3 x 10(6) vs IL-2 -/-, 20.9 x 10(6)). The latter also bear activation markers. In addition, thymocytes from TNP-KLH-immunized IL-2 -/- mice produce more IFN-gamma and less IL-4 than similarly immunized IL-2 +/+ mice. These defects in thymocyte maturation and lymphokine production are IL-12 driven, since they are prevented when immunized IL-2 -/- mice are coadministered with anti-lL-12. Furthermore, we demonstrate that IL-2 -/- mice exhibit decreased cortical apoptosis as determined by thymocyte numbers and detection of apoptotic cells in situ. Finally, we show that colitis-inducing thymocytes are generated in the immunized IL-2 -/- thymus, since IL-2 +/+ mice develop colitis following injection of small numbers of single positive thymocytes from immunized IL-2 -/- mice but not from IL-2 +/+ mice. Taken together, these data indicate that, in the absence of IL-2, thymocyte maturation is abnormally directed by IL-12 toward the generation of mature, activated Th1-type thymocytes that are capable of mediating colitis.
Cognate T cell help for CD40-deficient B cells induces c-myc RNA expression, but DNA synthesis requires an additional signal through surface Ig.
Authors: Schrader C E , Stavnezer J , Kikutani H , Parker D C
Published: December 2022

To investigate the role of CD40 ligand in the delivery of help to B cells, we examined the Ag-specific interaction of B cells from CD40-deficient mice with a Th2 cell line in vitro. Small resting B cells from normal mice are stimulated to synthesize DNA when they present monovalent Ag (rabbit Fab anti-Ig) to a rabbit Ig-specific Th cell line. This response, which is independent of a signal through the B cell Ag receptor (sIg), is nearly absent in B cells from CD40-deficient mice. The CD40-deficient B cells are not defective in Ag presentation because they induce T cell IL-4 synthesis as well as normal B cells. Also, CD40-deficient B cells respond to T cell help with DNA synthesis almost as well as normal B cells if an additional signal is provided through sIg. In conjunction with a sIg signal, cell contact with helper T cells induces DNA synthesis more effectively than soluble cytokines. CD40-independent T cell help can also be measured as an early increase in c-myc mRNA levels in CD40-deficient B cells presenting Ag to helper T cells, although the levels of c-myc RNA expression are lower than those in normal B cells. However, c-myc RNA induced by noncognate interaction with anti-CD3-activated T cells is completely CD40 dependent. We conclude that early growth signals from activated Th cells are received by CD40-/- B cells, but that CD40 and/or sIg signals are required for efficient induction of DNA synthesis.
B cell epitope mapping of the bacterial superantigen staphylococcal enterotoxin B: the dominant epitope region recognized by intravenous IgG.
Authors: Nishi J I , Kanekura S , Takei S , Kitajima I , Nakajima T , Wahid M R , Masuda K , Yoshinaga M , Maruyama I , Miyata K
Published: December 2022

We showed that i.v. IgG contains Abs against a major group of bacterial superantigens, and that they can inhibit superantigen-elicited T cell activation. The B cell epitope region of the superantigen and the inhibitory mechanism have remained unknown. To analyze the dominant B cell epitopes on the bacterial superantigen SEB (staphylococcal enterotoxin B), we constructed fusion proteins of SEB deletion mutants, and the reactivities of these recombinant proteins to i.v. IgG and healthy human sera were evaluated by means of immunoblotting. Intravenous IgG and healthy human sera mostly recognized the C-terminal fragment (amino acid (aa) 133-239). The C-terminally truncated protein (aa 1-228) and the truncated mutant delta 225-234 lost reactivity, while the truncated protein (aa 1-234) did not, suggesting that the region (aa 225-234) is the dominant B cell epitope. The mutant, in which residues 226-229 of SEB were exchanged for residues 209-212 of streptococcal pyrogenic exotoxin A, reduced the reactivity with the C-terminal region-specific IgG purified by affinity chromatography. The C-terminal region-specific IgG inhibited SEB-elicited T cell activation, suggesting that this Ab that recognizes the epitope functions as the humoral defensive factor against SEB in humans. Furthermore, the assumed epitope region was homology to the residues (aa 32-41) of human thymopoietin, containing the biologic active site.
Characterization of lymphocyte function-associated antigen 1 (LFA-1)-deficient T cell lines: the alphaL and beta2 subunits are interdependent for cell surface expression.
Authors: Weber K S , York M R , Springer T A , Klickstein L B
Published: December 2022

The leukocyte, or beta2, integrins are heterodimeric cell surface molecules that share a common beta subunit, and have unique alpha subunits. LFA-1 is the predominant beta2 integrin on lymphocytes, and plays an important role in many lymphocyte functions; however, most studies of the cytoplasmic regions of LFA-1 have been performed in transfected epithelial cells, such as COS, in part because no lymphoid cell lines deficient in the LFA-1 alpha subunit have been described. To address structure-function studies of beta2 integrins in relevant cell types, two T lymphoblastoid cell clones that completely lack cell surface LFA-1, J-(beta2).7 and SK-(beta2).7, derived from Jurkat and SKW3, respectively, were prepared by chemical mutagenesis and selection. Biosynthetic labeling and immunoprecipitation showed that the J-(beta2).7 clone did not translate any LFA-1 alpha subunit protein, while the SK-(beta2).7 cells did not synthesize any beta2 subunit protein. Northern blot analysis of poly(A+) RNA from these cells revealed an absence of the corresponding mRNA in each case. By transfection analysis, only the alpha subunit reconstituted LFA-1 expression in the J-(beta2).7 cells, while only the beta subunit restored cell surface LFA-1 expression in the SK-(beta2).7 cells. Functional studies with the parental cell lines, the J-(beta2).7 and SK-(beta2).7 cells, and the transfectants showed that all binding of Jurkat and SKW3 cells to purified ICAM-1 is mediated by LFA-1, and the reconstituted LFA-1 expressed by the J-(beta2).7 and SK-(beta2).7 transfected cells is regulated normally.
Immune CD8+ T lymphocytes lyse Listeria monocytogenes-infected hepatocytes by a classical MHC class I-restricted mechanism.
Authors: Jiang X , Gregory S H , Wing E J
Published: December 2022

Hepatocytes constitute the principal site of listerial replication in the livers of mice infected i.v. CD8+ T lymphocytes play a predominant role in the host defenses to Listeria monocytogenes. In vitro experiments by others undertaken to delineate the functions of CD8+ T lymphocytes have focused primarily on their interaction with Listeria-infected macrophages. Such experiments do not address directly the role of CD8+ T lymphocytes in eliminating the bulk of Listeria replicating within the liver. Here, we report that immune CD8+ T cells at an E:T cell ratio > or = 10:1 lysed Listeria-infected hepatocytes as judged by the following two criteria. Aspartate aminotransferase activity in the culture supernatants, indicative of hepatocyte damage, increased significantly. Conversely, infected hepatocytes cocultured with immune CD8+ T cells exhibited a marked reduction in viable intracellular Listeria assessed by CFUs. Neither immune CD4+ T cells nor nonimmune CD8+ T cells caused a similar increase in aspartate aminotransferase activity released or a decrease in intracellular bacteria. Immune CD8+ T cell-mediated lysis of infected hepatocytes was restricted by classical MHC class I (H-2Kb) molecules and was inhibited by the presence of either brefeldin A or mAb specific for CD8. These results suggest that the predominant role of CD8+ T lymphocytes in host resistance to listerial infections of the liver may be due to their capacity to lyse infected hepatocytes.
Albumin nitrosylated by activated macrophages possesses antiparasitic effects neutralized by anti-NO-acetylated-cysteine antibodies.
Authors: Mnaimneh S , Geffard M , Veyret B , Vincendeau P
Published: December 2022

Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi. This effect is not observed in the absence of albumin in the culture medium but is restored by the addition of albumin, indicating the presence of an albumin-nitric oxide (NO) adduct acting as an effector molecule. Since L-cysteine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-BSA conjugate. This conjugate was nitrosylated using sodium nitrite as a NO donor. Binding of NO to the conjugated haptens was assayed using spectrophotometry. It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond. Polyclonal Abs were obtained after immunizing rabbits with S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates. Using the enzyme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated compounds and other nitrosylated or non-nitrosylated compounds. The resulting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound. These Ab were used for an in vitro study of the kinetics of NO-derived compounds from activated murine macrophages. Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activated macrophages on the extracellular parasite, T. musculi. Moreover, the L-arginine-dependent antiparasitic activity of supernatants from Calmette-Guerin bacillus-activated macrophages required the presence of albumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effector role of S-nitroso-albumin.
IL-6 produced by macrophages infected with Mycobacterium species suppresses T cell responses.
Authors: VanHeyningen T K , Collins H L , Russell D G
Published: December 2022

The ability of Mycobacterium bovis Calmette-Guérin bacillus-infected bone marrow-derived macrophages to process and present exogenously added Ags to T cells and stimulate their growth and production of IL-2 was examined. The infected macrophages were inhibited in their ability to activate T cells, and this inhibition could be transferred to uninfected macrophages with filtered supernatants from mycobacteria-infected macrophages. The inhibition was not due to decreases in macrophage viability, Ag uptake, or cell surface expression of MHC class II or other accessory molecules necessary for Ag presentation. Other intracellular pathogens such as Listeria monocytogenes and Leishmania mexicana did not induce the soluble inhibitory factor, while Mycobacterium avium strain 101 did, suggesting the factor is specific to infection with mycobacteria. The inhibitory effect was reversed completely by preincubation with neutralizing Abs against IL-6, and rIL-6 partially restored the effect. Approximately 10,000-fold more IL-6 was produced by mycobacteria-infected macrophages compared with uninfected controls. Such sustained levels of IL-6 may account for the immune unresponsiveness apparent in both human and murine mycobacterial disease.
B-1 cell (CD5+B220+) outgrowth in murine schistosomiasis is genetically restricted and is largely due to activation by polylactosamine sugars.
Authors: Velupillai P , Secor W E , Horauf A M , Harn D A
Published: December 2022

Previously, we demonstrated that lacto-N-fucopentaose III, a sugar found on egg Ags of Schistosoma mansoni, stimulated splenic B cells from parasite-infected mice to proliferate and produce IL-10 and PGE2. The major source of B cell IL-10 is the B-1 subset (CD5+B220+). Thus we examined whether levels of peritoneal exudate B-1 cells changed as a consequence of infection. In CBA/J, BALB/c, and C3H/HeJ mice, we observed significant increases in B-1 cells at 2 to 4 wk postinfection, declining to baseline by 6 to 8 wk. In contrast, the percentage of B-1 cells remained unchanged in C57BL/6 and BALB/c X.id mice after infection. B-1 cells were not observed in the spleens of infected mice; however, coincident with peritoneal B-1 cell decline, splenic CD23+B220+ cells increased from 11% to 30%. Peritoneal B-1 cells could also be expanded by injection of soluble egg Ag, but not by its deglycosylated form, suggesting a role for carbohydrates in B-1 recruitment. In addition, these cells secreted in vitro large amounts of IL-10 in response to lacto-N-fucopentaose III. Further, this sugar induced B-1 cell outgrowth in CBA/J and C3H/HeJ mice, but not in C57BL/6 mice. Thus, early activation of polylactosamine-reactive, IL-10-producing peritoneal B-1 and splenic B cells may be related to early dominance of the Th2-type CD4+ T cell subset. The degree to which this occurs may in part explain differences in the degree of granulomatous pathology reported among various strains of mouse.