bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of IL-12 p40 messenger RNA expression and IL-12 production of macrophages via CD40-CD40 ligand interaction.
Authors: Kato T , Hakamada R , Yamane H , Nariuchi H
Published: December 2022

The mechanism of IL-12 production has been studied by stimulating macrophages or B cell lines with LPS, Staphylococcus aureus, or phorbol diester. However, since IL-12 plays an important role in the activation of T cells interacting with APC, it is important to study the mechanism of IL-12 production induced by T helper cell-APC interaction. We and others have demonstrated that IL-12 is produced in cultures where Th1 cells are stimulated with Ag or APC. In the present experiments, we studied a role of CD40-CD40 ligand (CD40L) interaction in IL-12 production and obtained the following results: 1) incubation of normal Th1 clone with APC in the presence of Ag induced IL-12 p40 and p35 mRNA accumulation and IL-12 production, and the addition of anti-CD40L blocked the p40 mRNA accumulation and IL-12 production but not p35 mRNA accumulation; 2) when Th1 clone from a CD40L-deficient mouse was used in the incubation, p35 mRNA accumulation was induced, but neither p40 mRNA accumulation nor IL-12 production was induced; 3) CD40L+ Th1 clone, or insect cell membrane expressing mouse CD40L, induced p40 mRNA accumulation and IL-12 production but not p35 mRNA accumulation. These results indicate that the CD40-CD40L interaction plays a critical role in IL-12 p40 mRNA accumulation and bioactive IL-12 production and that p35 mRNA accumulation was regulated via a different mechanism than CD40-CD40L interaction. Most of the cells producing IL-12 were Mac-1+ macrophages.
Intact antigen receptor-mediated calcium signals in patients with early stage HIV-1 infection.
Authors: Hickey T E , Blair P J , Perfetto S , Smoot D S , Wagner K F , St Louis D C , Mayers D L , Siegel J N , June C H
Published: December 2022

A variety of deficiencies in T cell activation have been described in HIV-1 infection. To determine whether one component of Ag receptor signal transduction might be impaired and contribute to the immunopathology of HIV infection, we tested CD4 cells from patients with early to mid-stage HIV infection for TCR-induced calcium mobilization. There was no detectable difference between patients and controls in the mean CD4 cell calcium response or in the fraction of responding CD4 cells after cross-linking the TCR with OKT3 Ab. In addition, in HIV-infected patients, there was no correlation between calcium mobilization and the CD4 cell count. These results indicate that there are no intrinsic impairments of Ag receptor calcium signaling in circulating CD4 cells from HIV-infected patients with more than 400 CD4 cells/mm3, although abnormalities in patients with later stage infections cannot be excluded.
Modulation of antigen presentation and class II expression by a class II-associated invariant chain peptide.
Authors: Zechel M A , Chaturvedi P , Lee-Chan E C , Rider B J , Singh B
Published: December 2022

During the process of MHC class II assembly, the class II-associated invariant chain peptide (CLIP) remains bound within the peptide binding groove until its subsequent removal, which is mediated by H-2 M. We have defined the functional role of CLIP, through saturation of the endosomal compartment with exogenous CLIP-(85-101), resulting in reduced class II MHC on the surface of APCs and an impeded T cell response. Conversely, incubation of the same cells with immunogenic peptides or proteins resulted in an up-regulation of surface class II MHC. T cells from CLIP- plus Ag-immunized mice showed a marked decrease in Ag-specific response over that in mice primed with Ag alone. A B cell hybridoma, TA3 (H-2d,k) incubated with CLIP in vitro showed dramatically reduced MHC class II I-A surface expression. APCs derived from CLIP-immunized mice exhibited down-regulation of surface class II MHC, but not of CD45 (B220). Electrophoretic studies showed that the addition of exogenous CLIP resulted in a relative decrease in SDS-stable MHC class II heterodimers in TA3 cells. Studies with FITC-CLIP and FITC-OVA-(323-339) peptides demonstrated that exogenously added CLIP peptide does not bind to surface class II molecules via the endogenous route, whereas OVA peptide does. This suggests that exogenously added CLIP acts intracellularly, perhaps in the compartment where H-2 M intersects with class II molecules. These findings demonstrate the functional role of CLIP to regulate MHC class II-mediated Ag presentation in CD4+ T cell responses.
Studies of tum- peptide analogs define an alternative anchor that can be utilized by Ld ligands lacking the consensus P2 anchor.
Authors: Robinson R A , Lee D R
Published: December 2022

To determine how peptides that lack a consensus binding motif interact with class I molecules, we have studied the binding of the tumor-associated tum- P91A 14-22 (tum-) peptide to Ld. Previously, a proline at position 2 (P2) and a hydrophobic residue at P9 had been defined as anchors for Ld ligands. However, the tum- peptide lacks the P2 proline anchor. To compare how peptides with and without the P2 proline anchor bind to Ld, we analyzed the binding of monosubstituted analogues of the murine cytomegalovirus (MCMV) pp89 168-176 and the tum- peptides to Ld. As expected, the binding of both peptides was inhibited by substitutions at P9, the carboxyl-terminal anchor. As also predicted, the MCMV peptide was found to be dependent upon its P2 proline for binding to Ld. By contrast, the binding of the tum- peptide to Ld is dependent primarily on a P8 aspartate residue. Interestingly, the p2Ca peptide that is immunodominant in allorecognition of Ld also lacks the P2 proline anchor and has been shown to depend on residues near the carboxyl terminus for binding to Ld. Furthermore, both the p2Ca and the tum- peptides can bind to Ld as octamers. These combined studies suggest that there are at least two alternative manners by which peptides can bind to Ld. Although most Ld ligands bind using a P2 proline anchor, the tum- and p2Ca peptides bind using alternative anchors in the carboxyl-terminal region.
Neutralizing antibody responses elicited in mice immunized with recombinant bacillus Calmette-Guérin producing the Schistosoma mansoni glutathione S-transferase.
Authors: Kremer L , Riveau G , Baulard A , Capron A , Locht C
Published: December 2022

Schistosomiasis is a group of severe parasitic diseases, in humans and domestic animals, that are especially of importance in the developing world. No efficacious vaccine is currently available. However, Ab-mediated immune responses against the 28-kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST) appear to be involved in protection. This Ag was produced in recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG). The recombinant protein bound glutathione and expressed enzymatic activity, indicating that the active site of Sm28GST was folded properly. Single i.v., i.p., s.c., or intranasal immunizations with rBCG in BALB/c mice resulted in significant anti-SM28GST Ab responses, which were enhanced by a booster dose. The Ab responses remained high for at least 1 yr after immunization. Analyses of the isotype profiles indicated that i.v. immunized mice produced high titers of anti-Sm28GST IgG2a, and less IgG2b and IgG1. Mice immunized by the s.c. route initially also produced high levels of IgG2a and low titers of IgG1 and IgG2b, but the titers of the latter two isotypes increased gradually thereafter, tending toward a mixed profile. Intraperitoneal immunization provided a mixed profile directly after the first administration. High titers of anti-Sm28GST Abs also corresponded to high levels of neutralization of the enzymatic activity. These results indicate that rBCG induces strong IgG1, IgG2a, and IgG2b, and neutralizing Ab responses against Sm28GST, which has been found to correlate with protection against S. mansoni in humans.
Genetically determined differences in IL-10 and IFN-gamma responses correlate with clearance of Chlamydia trachomatis mouse pneumonitis infection.
Authors: Yang X , HayGlass K T , Brunham R C
Published: December 2022

Cytokine patterns elicited by infection are critical in the regulation of the adaptive immune response and in the resolution of infection. Using a murine model of pneumonia induced by intranasal inoculation with the Chlamydia trachomatis mouse pneumonitis (MoPn) biovar, we found that the patterns of immune responses and cytokine production by spleen cells were correlated with quantitative growth of MoPn in the lungs of C57BL/6 and BALB/c mice. Specifically, BALB/c (H-2d) mice had a significantly higher mortality rate and a slower clearance of the organism from the lungs than did C57BL/6 mice (H-2b). BALB/c mice exhibited higher IL-10 production, higher IgG1 Ab responses, and less IFN-gamma production than C57BL/6 mice, which showed higher IFN-gamma production, stronger delayed-type hypersensitivity (DTH) responses, and significantly less IL-10 production. In vivo neutralization of IL-10 in BALB/c mice with an anti-IL-10 mAB during MoPn infection significantly increased the DTH response and enhanced clearance of MoPn. These findings support the hypothesis that excessive IL-10 production in BALB/c mice inhibits Th1-like responses, including IFN-gamma expression and the DTH response following chlamydial infection and consequently delay infection resolution.
Autocrine activation of hemopoietic progenitor-derived myelo-monocytic cells by IFN-gamma gene transfer.
Authors: Ahuja S S , Brown M R , Fleisher T A , Ahuja S K , Malech H L
Published: December 2022

Immunomodulatory cytokines have been used with success as adjunctive therapy in genetic disorders such as chronic granulomatous disease and infectious diseases such as leishmaniasis and leprosy. As the first step toward developing novel methods to deliver immunomodulatory cytokines, we used retrovirus-mediated somatic gene transfer techniques to produce IFN-gamma from human peripheral blood CD34+ hemopoietic progenitor (PBHP) cells. After transduction, the PBHP cells were made to differentiate toward myelo-monocytic lineages. Only the PBHP-derived myelo-monocytic cells that were transduced with the IFN-gamma cDNA produced IFN-gamma(4 +/- 1.3 ng of IFN-gamma/10(6) PBHP cells.) Despite a reduction in the proliferation of IFN-gamma-transduced PBHP cells as well as a decrease in erythroid colony formation, there was an enhancement of monocyte differentiation and activation. Monocytes differentiated from the IFN-gamma-transduced PBHP cells demonstrated 1) up-regulation of MHC class I and II Ag expression, 2) increased Fc(gamma)RI expression, and 3) enhanced superoxide production in response to both opsonized zymosan (25-fold) and phorbol ester (3-fold). Furthermore, a functional response to a monocyte-specific chemokine, monocyte chemotactic protein-1 (mobilization of intracellular Ca2+) was seen only in the IFN-gamma-transduced cells. Thus, PBHP cells transduced with IFN-gamma cDNA produce not only biologically active IFN-gamma, but also enhanced monocyte differentiation, resulting in an activated state that includes unique functions, such as responsiveness to monocyte chemotactic protein-1. These transduced activated monocytes may be specifically suited to cellular therapy requiring homing to sites of inflammation where their anti- microbicidal, cytotoxic and APC functions play an important role in host defense against foreign pathogens.
Chemokine monocyte chemoattractant protein-1 is expressed by astrocytes after mechanical injury to the brain.
Authors: Glabinski A R , Balasingam V , Tani M , Kunkel S L , Strieter R M , Yong V W , Ransohoff R M
Published: December 2022

By 24 h after mechanical trauma to the cerebral cortex, astroglial reaction begins and injury sites are infiltrated by activated mononuclear phagocytes derived from blood-borne monocytes and endogenous microglia. There is little information about cellular interactions between astrocytes and leukocytes during this process. We previously showed that murine astrocytes produce chemokines including monocyte chemoattractant protein-1 (MCP-1) during experimental autoimmune encephalomyelitis. In this study, we asked whether astrocytes produce MCP-1 in the absence of immune mediated inflammation. To address this question, we analyzed the time course and cellular source of MCP-1 in mouse brain after penetrating mechanical injury, with particular focus on early time points before histologic detection of infiltrating mononuclear phagocytes. We observed sharply increased steady state levels of MCP-1 mRNA within 3 h after nitrocellulose membrane stab or implant injury to the adult mouse brain, and MCP-1 protein elevations were documented at 12 h postinjury. In situ hybridization combined with immunohistochemistry for the glial fibrillary acidic protein astrocyte marker showed that astrocytes were the cellular source of MCP-1 mRNA at these early time points after mechanical brain injury. Stab injury to the neonatal brain evoked neither MCP-1 expression nor astrogliosis. These results demonstrate that chemokine gene expression comprises one component of the astrocyte activation program. The data are consistent with a role for MCP-1 in the central nervous system inflammatory response to trauma.
Membrane cofactor protein (MCP; CD46). Isoforms differ in protection against the classical pathway of complement.
Authors: Liszewski M K , Atkinson J P
Published: December 2022

Membrane cofactor protein (MCP; CD46) is a widely distributed C3b/C4b-binding glycoprotein that inhibits complement activation on host cells. MCP is expressed primarily as four isoforms that arise by alternative splicing of a single gene. The differences reside in the domains for O-glycosylation and cytoplasmic tails. Tissue-specific expression of isoforms and the differential processing of precursors mediated by the cytoplasmic tails suggest that isoform variations are biologically significant. The goal of these experiments was to characterize the complement inhibitory profile of the four commonly expressed isoforms. The MCP isoforms (BC) with a larger O-glycosylation domain bound C4b more efficiently than the C isoforms, which are smaller and less glycosylated in this region. Additionally, cytoprotection assays of individual clones of transfected isoforms bearing equivalent copy numbers demonstrated that the BC isoforms also provided enhanced protection in a classical pathway-mediated system and cleaved cell-bound C4b more efficiently than the C isoforms. Taken together, these data demonstrate that BC isoforms preferentially protect against the classical pathway of complement. Such findings indicate a physiologic role for isoform variation and have therapeutic implications for use of MCP isoforms as complement inhibitors in such areas as xenotransplantation.
Binding of model soluble immune complexes to modified C-reactive protein.
Authors: Motie M , Brockmeier S , Potempa L A
Published: December 2022

We have identified a binding interaction between a modified form of C-reactive protein (mCRP) and model immune complexes. We have characterized these interactions in terms of their relative affinity, specificity, pH dependence, and recognition site on mCRP. First, binding isotherms were generated for the reaction of immobilized mCRP with heat-aggregated IgG (HAG) which gave an estimate for the value of the dissociation constant, Kd, of 1.6 nM. Second, competitive binding assays were performed using immobilized HAG to determine the relative affinity (IC50) for the interaction between HAG or monomeric IgG and mCRP in the fluid phase. While the binding of HAG to mCRP occurred with high affinity (IC50=0.72 nM), the binding of monomeric IgG to mCRP occurred with >2000-fold lower affinity (IC50>1.66 muM). Third, immune complex binding to immobilized mCRP was studied using defined ratios of horseradish peroxidase and rabbit anti-peroxidase Ab. The binding of these complexes to mCRP was strongly pH dependent, with a maximum at pH 5.5. At this optimal pH, preformed peroxidase-antiperoxidase immune complexes (approximately 2:3 Ab:Ag molar ratio) were shown to bind immobilized mCRP with a Kd of 111 nM. Fourth, using mCRP-specific mAbs, HAG and peroxidase- antiperoxidase binding sites were localized to CRP sequences to 130 to 138 and 200 to 206. Since inflammatory processes often cause microenvironments to become acidic, and since mCRP selectively binds immune complexes at acidic pH, we propose that mCRP mediates the specific binding of immune complexes in vivo and potentiates effector reactions for immune complex removal.
Late induction of CREB/ATF binding and a concomitant increase in cAMP levels in T and B lymphocytes stimulated via the antigen receptor.
Authors: Feuerstein N , Firestein R , Aiyar N , He X , Murasko D , Cristofalo V
Published: December 2022

Transcription factors of the cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family were implicated in the expression of T cell-specific genes and in the expression of oncogenic retroviruses associated with leukemia in T and B lymphocytes. To study the regulation of CREB/ATF transcription factors during lymphocyte activation, studies were pursued in primary cultures of resting murine splenic T and B lymphocytes stimulated via the Ag receptor. Using consensus/CRE and proliferating cell nuclear Ag (PCNA)/CRE as probes in the DNA binding assay, we showed that a marked induction of CRE binding is associated with activation of splenic T lymphocytes with anti-CD3 Ab. CRE binding was markedly induced after 48 h; it gradually declined at 72 h, but remained elevated above control levels after 120 h. Most significant, activation by anti-CD3 was associated with a marked induction of cAMP levels that preceded the onset of DNA synthesis and the induction of IL-2 secretion and reached a peak after 48 h (9.5- to 11-fold), concomitant with the peak in CRE binding. Rapamycin, a potent immunosuppressant, inhibited the induction of cAMP levels by anti-CD3 concomitant with inhibition of CRE binding activity and arrest of DNA synthesis. A marked induction in CRE binding after 48 h was also found in splenic B lymphocytes stimulated by LPS and anti-Ig and was correlated with a 3- to 4-fold increase in the intracellular levels of cAMP. Two inducible CRE complexes were found to bind to consensus/CRE and PCNA/CRE; the major complex contained primarily CREB homodimers and was constitutively expressed in resting lymphocytes. Conversely, stimulation of lymphocytes was associated with formation of a new, slow migrating CRE complex that demonstrated high inducibility in both consensus/CRE and PCNA/CRE. We show that this de novo inducible CRE complex contains CREB and ATF2, but not ATF1. Taken collectively, these results suggest that recruitment of CREB and ATF2 to the promoter of genes is tightly regulated during activation of T and B lymphocytes and implicate a cross-talk of cAMP and non-cAMP pathways in the regulation of transcriptional processes at late stages of activation in T and B lymphocytes stimulated via the Ag receptor.
Murine NK cell allospecificity-1 is defined by inhibitory ligands.
Authors: Zheng W P , Kiura K , Milisauskas V K , DeNardin E , Nakamura I
Published: December 2022

Hemopoietic allografts of normal and neoplastic origin are subject to NK cell-mediated resistance in mice. Susceptibility to this resistance is controlled by MHC-linked genes in a recessive manner. Several distinct specificities could be postulated to explain the strain-dependent pattern of resistance. These presumptive specificities for recognition are H-2 haplotype dependent, but the correspondence is not one-to-one. For example, resistance of H-2d or H-2b/d host to H-2 b graft operationally defines specificity-1, establishing its link with haplotype H-2b. To examine the molecular basis of specificity-1, spontaneous Dd-loss mutant clones were isolated from H-2b/d and H-2d hemopoietic cell lines, i.e., 416B of (C57BL/6 x DBA/2)F1 (B6D2F1) origin and L1210 of DBA/2 origin, both of which lack specificity-1. The expression of specificity-1 in the mutant clones was examined in vivo and in vitro. The results indicate that Dd-loss clones of 416B and L1210 lines express specificity-1. These data suggest that murine NK cell allospecificity-1 is defined primarily by the absence of the Dd molecule or other class I molecules sharing the protective motifs; no H-2b-associated genes play a relevant role. This conclusion is consistent with the missing self hypothesis of NK cell reactivity, and is in agreement with the observation that lysis of B6 targets by B6D2F1 NK cells is mediated mostly by cells that express Ly-49A and/or Ly-49G2.
Transport of anti-sperm monoclonal IgA and IgG into murine male and female genital tracts from blood. Effect of sex hormones.
Authors: Wang Y , Ben K , Cao X , Wang Y
Published: December 2022

Ab levels in the genital tract may be important in fertility and in preventing sexually transmitted diseases. In this study, 125I-labeled polymer or monomer mAb IgA (C4pIgA or C4mIgA) and IgG2b (C4IgG) to murine lactate dehydrogenase C4 and a polymer mAb IgA (nplgA) not cross-reacting with mouse sperm were intravenously injected into BALB/c mice, and the relative distribution of these Abs was determined. Polymer IgA was transported much more efficiently into the genital tract, trachea, and duodenum of both sexes than C4IgG and C4 mIgA (p < 0.01). The transport of polymer IgA (C4pIgA and npIgA) into the male genital tract greatly increased following orchiectomy (p < 0.01); this change was not affected by testosterone, suggesting that the unknown regulatory factor(s) from the testis may suppress polymer IgA transport. However, the transport of polymer IgA into female genital tissues was significantly decreased by ovariectomy (p < 0.01); this decline can be rectified by beta-estradiol but not progesterone treatment, suggesting that estradiol may stimulate polymer IgA transport. Furthermore, the transport of C4IgG into tissues of the Fallopian tubes and the uterus was significantly decreased by treatment with progesterone (p < 0.01). Together, these findings indicate that serum polymer IgA can be transported selectively into the genital tracts of both sexes, that this transport is strongly under the control of gonads, and that transport of IgG into the Fallopian tubes and uterus is down-regulated by progesterone.
Intervention of CD4+ cell subset shifts and autoimmunity in the BXSB mouse by murine CTLA4Ig.
Authors: Chu E B , Hobbs M V , Wilson C B , Romball C G , Linsley P S , Weigle W O
Published: December 2022

In the BXSB autoimmune disease-prone mouse strain, male mice develop severe lupus-like symptoms and die early in life (4-6 mo), whereas females do not. We have previously demonstrated that profound phenotypic and functional changes occur with age in CD4+ cells from BXSB males. CD4+ cells from males (4 mo old) were predominantly CD44high, CD45RBlow, and MEL-14low (activated/memory phenotypes), while the reciprocal phenotypes characteristic of naive cells were prevalent in age-matched females and young adult males (2 mo old). CD4+ cells from older males proliferated less and produced less IL-2 and IFN-gamma than cells from either females or young males in response to immobilized anti-CD3 mAb. We tested the effect of CTLA4Ig treatment on the progression of disease in BXSB males. CD4+ cells from CTLA4Ig-treated mice at 4 mo of age were predominantly CD44low, CD45RBhigh, and MEL-14high phenotypes that were identical with those observed in CD4+ cells from young (3-mo-old) females. In contrast, control male mice treated with IgG2a accumulated the CD4+ memory phenotype. CD4+ cells from 4-mo-old male CTLA4Ig-treated mice proliferated and produced IL-2 at levels similar to those of cells from females in response to immobilized anti-CD3 mAb. Furthermore, in contrast to IgG2a-treated mice, female and CTLA4Ig-treated male mice at 4 mo of age produced no anti-chromatin Abs. Three of four male mice injected with CTLA4Ig until 6 mo of age appeared healthy at 8 mo of age, whereas all five of IgG2a-treated control males died by 6 mo of age. These 8-mo-old CTLA4Ig-treated males showed variable resistance to autoimmunity as well as function and phenotype marker expression, and a less striking glomerulonephritis than 4-mo-old untreated males. The results of this study demonstrate that the rampant T cell activation and T cell dysfunction that occur in male BXSB mice by 4 mo of age are abrogated by blocking the CTLA4-dependent costimulatory signal(s). They also show that treatment with CTLA4Ig can suppress the pathogenesis of disease and increase longevity.
Oral antigen inhibits priming of CD8+ CTL, CD4+ T cells, and antibody responses while activating CD8+ suppressor T cells.
Authors: Ke Y , Kapp J A
Published: December 2022

We have been investigating the mechanisms by which exogenous protein Ags activate CD8+ T cells. Previously, we have shown that OVA primed CTL precursors in vivo if administered as an emulsion with an adjuvant such as CFA. Such CTLs inhibit development of Ab responses when adoptively transferred into syngeneic mice. Thus, CD8+ CTL are immunosuppressive. These studies were initiated to determine whether CD8+ suppressor T cells were cytolytic T cells. Oral administration of protein Ags is a well-established method for inducing tolerance in humoral and delayed hypersensitivity responses which is associated with development of CD8+ suppressor T cells. OVA was chosen as a model Ag because it has been used extensively in oral tolerance studies, and target cells expressing the OVA gene are well characterized. Our data show that multiple, intragastric doses of native OVA inhibited priming of CD8+ CTL precursors and also inhibited CD4+ T cells and Ab responses. Oral OVA inhibited CTL priming in mice immunized with OVA-loaded EL4 cells, OVA-expressing transfectants, or OVA in CFA. The observed tolerance is specific to the orally administered Ag. Although oral OVA did not prime CTL precursors, it did activate spleen cells that transferred unresponsiveness to naive syngeneic mice. Suppression was mediated by CD4-CD8+ T cells. These CD8+ suppressor T cells were phenotypically distinguished from CTL by reactivity with a mAb that recognizes activated suppressor T cells.
Long-term culture of triple-negative thymocytes.
Authors: Montecino-Rodriguez E , Dorshkind K
Published: December 2022

The role of the thymic microenvironment in regulating events that occur subsequent to the acquisition of CD4, CD8, and the TCR, such as positive and negative selection, has been studied extensively. However, comparatively less is known about how thymic stromal cells regulate growth and differentiation within the CD4-CD8-TCR- triple negative (TN) cell compartment. Long-term culture systems have played a pivotal role in the understanding of microenvironmental regulation of myelopoiesis and B lymphopoiesis, but establishment of comparable cultures for T lineage cells has proved challenging. This report describes a culture system in which a thymic stromal cell line preferentially supports the long-term growth of TN thymocytes. In addition to demonstrating that TN cells have a considerable proliferative potential, the results provide insights into the effects of IL-7 on the growth and/or survival of TN cells and its role in regulating TCR gene rearrangements. The ability of the cultured TN cells to mature into CD4- and/or CD8-expressing thymocytes, some of which express TCR-alpha beta, suggests that the system will be valuable for the identification of microenvironmental stimuli that regulate the maturation of TN cells.
Acquisition of surface IgD fails to protect from tolerance-induction. Both surface IgM- and surface IgD-mediated signals induce apoptosis of immature murine B lymphocytes.
Authors: Norvell A , Monroe J G
Published: December 2022

While mature splenic B cells are surface (s)IgM+ and sIgD+, immature, tolerance-susceptible B cells express sIgM and varying levels of sIgD. Differential expression of sIgD on tolerance-susceptible and resistant B cells suggests that sIgM and sIgD may transmit qualitatively different signals. Alternatively, tolerance sensitivity may be associated with intrinsic differences in sIg signaling, regardless of the isotype engaged. Here, we have exploited a stage of B cell development at which immature, tolerance-sensitive B cells co-express sIgD and sIgM. Using these immature stage B cells to evaluate isotypic differences in the ability to induce activation and deletion, we have found that neither ligation of sIgD nor sIgM is capable of inducing proliferation. Moreover, in contrast to mature B cells, both sIgD and sIgM induce apoptosis by immature stage B cells. Importantly, cross-linking sIgD does not protect immature B cells from sIgM-induced apoptosis. Thus, the differences in tolerance susceptibility of immature and mature B cells must be due to intrinsic developmental rather than isotypic differences in Ag receptor signal transduction.
IL-12 expression in AIDS-related lymphoma B cell lines.
Authors: Benjamin D , Sharma V , Kubin M , Klein J L , Sartori A , Holliday J , Trinchieri G
Published: December 2022

IL-12 is a 70-kDa heterodimer formed by the 40-kDa heavy chain (p40) and the 35-kDa light chain (p35). Twenty-five Burkitt's lymphoma cell lines (CL) and seven normal lymphoblastoid B CL were studied. The Burkitt's CL included AIDS-associated B CL (AABCL) (7 EBV+/2 EBV-) and non-AABCL (8 EBV+/8 EBV-). Reverse transcription-PCR detected p40 in EBV+ AABCL (7 of 7), EBV+ non-AABCL (3 of 8), and normal lymphoblastoid B CL (6 of 6) but not in EBV- CL (0 of 10). p35 mRNA was detected in 30 of 30 CL. Constitutive secretion of p40 was found in 7 of 7 EBV+ AABCL (range, 341-18,086 pg/ml) and p70 in 3 of 7 EBV+ AABCL (range, 25-197 pg/ml), but in only 1 of 8 EBV+ non-AABCL and 0 of 7 normal lymphoblastoid CL. PMA stimulated p40 secretion in 7 of 7 EBV+ AABCL and p70 secretion in 5 of 7 EBV+ AABCL. PMA also triggered p40 and p70 secretion in 2 EBV+ non-AABCL and in 3 of 7 normal lymphoblastoid CL. No IL-12 secretion was detected in 10 EBV- CL, including EBV- AABCL. The CL produced IL-10, a known inhibitor of IL-12, but anti-IL-10 Abs did not neutralize IL-12. Similarly, neutralizing anti-IFN gamma Abs or IFN gamma did not affect B cell IL-12. For IL-12R studies, reverse transcription-PCR and 125I-IL-12 binding assays were performed. Although all CL tested showed mRNA accumulation for one of the IL-12R components, IL-12 binding sites were detected in only 1 of 30 CL. Our data suggest that: 1) AABCL constitutively secrete large amounts of IL-12, contrasting with low IL-12 production by HIV-1 infected PBMC; 2) lack of IL-12 expression in EBV- AABCL suggests that in vivo exposure of B cells to HIV-1 only does not induce IL-12 secretion and that both HIV-1 and EBV are required; 3) the autocrine-negative effect of IL-10 on IL-12 in monocytes and the enhancing effect of IFN gamma on IL-12 secretion do not apply to B cells derived from AIDS patients.
Neutralization of IL-12 in vivo prevents induction of contact hypersensitivity and induces hapten-specific tolerance.
Authors: Riemann H , Schwarz A , Grabbe S , Aragane Y , Luger T A , Wysocka M , Kubin M , Trinchieri G , Schwarz T
Published: December 2022

IL-12 is a heterodimeric cytokine with a powerful positive regulatory influence on the development of Th1 cell-mediated immune responses. Th1 cells are critically involved in contact hypersensitivity (CHS), so we were interested in studying whether IL-12 is of relevance in the induction phase of CHS. BALB/c mice were sensitized by epicutaneous application of 0.5% 2,4-dinitrofluorobenzene (DNFB) on the abdomen, draining lymph nodes obtained at various time points thereafter. RNA was extracted, and PCR analysis was performed using primers specific for the IL-12 subunits p35 and p40. Although p35 was constitutively expressed and not affected by hapten application, p40 transcripts were found to be enhanced in lymph nodes obtained between 12 and 24 h after sensitization. To study whether IL-12 is functionally relevant for the induction of CHS, an mAb directed against recombinant murine IL-12 was injected i.p. 3 and 24 h before sensitization. Challenge was performed 5 days later by painting the left ear with 0.3% DNFB. Whereas mice injected with sodium chloride or an isotype control Ab responded with a normal ear swelling after challenge with DNFB, mice treated with anti-IL-12 Ab showed a significantly reduced ear swelling response. To study whether injection of the IL-12 Ab causes transient nonresponsiveness or tolerance, resensitization was performed after a resting period of 14 days. Groups of mice initially treated with the anti-IL-12 Ab plus DNFB showed only a minimal response to DNFB after resensitization, suggesting that these mice became tolerant. Tolerance appeared to be hapten-specific because these mice could be successfully sensitized with the nonrelated hapten 2,4,6-trinitrochlorobenzene. Furthermore, when the anti-IL-12 Ab was injected into already sensitized mice before challenge, ear swelling response was significantly suppressed, suggesting that IL-12 also is involved in the effector phase of CHS. To exclude the possibility that the inhibitory effect of the anti-IL-12 Ab is simply due to an anti-inflammatory activity of the Ab, the anti-IL-12 Ab was injected i.p. 3 and 24 h before epicutaneous application of the toxic compound croton oil. However, irritant dermatitis elicited by croton oil was not affected by the Ab. Thus, the present study demonstrates that in vivo application of an anti-IL-12 Ab inhibits sensitization and induces hapten-specific tolerance and thus suggests that IL-12 may play a dominant in vivo role in the induction of CHS.
Suppression of T cell proliferation by tumor-induced granulocyte-macrophage progenitor cells producing transforming growth factor-beta and nitric oxide.
Authors: Young M R , Wright M A , Matthews J P , Malik I , Prechel M
Published: December 2022

Production of high levels of granulocyte-macrophage CSF (GM-CSF) by LLC-LN7 tumors results in myelopoietic stimulation and an increase in cells having natural suppressor (NS) activity. Prior studies showed these NS cells could be isolated from the bone marrow of tumor-bearing mice with an Ab (ER-MP12) that recognized GM-progenitor cells. The present study showed these cells to also be in the spleen, lymph node, and tumor, and that treatment of tumor-bearing mice with low doses of IFN-gamma plus TNF-alpha reduced the frequency of E-MP12+ cells. Studies focused on characterizing the intratumoral ER-MP12+ cells and the mechanism by which they suppress T cell proliferation. When isolated and seeded in soft agar with CSF-containing LLC-LN7 supernatants, the ER-MP12+ cells grew into colonies, most of which contained both granulocytic and monocytic cells. Tumor-derived ER-MP12+ cells and their culture supernatants were suppressive to T cell proliferation. Among the factors produced by ER-MP12+ cells were TGF-beta, nitric oxide (NO), IL-10, and prostaglandin E2 (PGE2). However, it was TGF-beta and NO that mediated the suppression of T cell proliferation by ER-MP12+ cells. Intratumoral ER-MP12+ cells could be maintained as suppressive blastlike cells for at least 4 days in cultures containing CSFs, but adding IFN-gamma plus TNF-alpha to these cultures caused their differentiation mainly into nonsuppressive TNF-alpha-secreting monocytic cells. These results show that intratumoral ER-MP12+ cells having homology to GM-progenitor cells suppress T cell function by producing TGF-beta and NO. IFN-gamma/TNF-alpha treatment stimulates their differentiation and shift from production of TGF-beta and NO to production of TNF-alpha.
Isolation and characterization of a unique natural killer cell inhibitory factor present in the anterior chamber of the eye.
Authors: Apte R S , Niederkorn J Y
Published: December 2022

The eye is a classic example of an immunologically privileged organ. Immune privilege in the anterior chamber is a multifaceted phenomenon that may have evolved to protect delicate ocular tissues from immune-mediated damage. The anterior chamber of the eye is lined with corneal endothelial cells, which are terminally differentiated cells that are incapable of regeneration. Since corneal endothelial cells are crucial for maintaining corneal clarity, damage to these cells leads to impairment of vision and, in some cases, blindness. The absence of constitutive expression of MHC class I molecules on corneal endothelial cells makes them potential targets for NK cell-mediated cytolysis. The aqueous humor (AH) that fills the anterior chamber of the eye and bathes the corneal endothelial surface contains numerous immunomodulatory molecules, such as TGF-beta, which may protect the corneal endothelium from NK cell-mediated damage. Functionally significant levels of TGF-beta are present in AH. In this study, we demonstrate that AH inhibits NK cell-mediated cytotoxicity in vitro, but does not affect cytotoxic T lymphocyte-mediated lysis. The inhibitory effect was immediate and occurred without preincubation of NK cells with aqueous humor. The inhibitory factor could not be neutralized by Abs to TGF-beta, but was abrogated by treating AH with proteases. Inhibition was not simply due to lysis of NK cells, since AH did not alter NK cell viability. Initial purification using gel filtration chromatography showed that the activity was restricted to a single elution peak. Analysis of the active fractions by gel electrophoresis indicated that the inhibitory factor was a single protein of approximately 10 kilodaltons. The NK inhibitory factor might be an important modulator for protecting the corneal endothelium from unbridled NK cell-mediated injury and, thus, might be instrumental in preserving vision.
Metabolism of gammalinolenic acid in human neutrophils.
Authors: Chilton-Lopez  , Surette M E , Swan D D , Fonteh A N , Johnson M M , Chilton F H
Published: December 2022

Gammalinolenic acid (GLA), when provided as a dietary supplement, has been reported to improve clinical symptoms of several inflammatory disorders. The goal of the current study was to examine the metabolism of GLA and its relationship to arachidonic acid (AA) in the human neutrophil. Initial studies indicated that neutrophils provided GLA in vitro rapidly elongate it (by two carbons) to dihomogammalinolenic acid (DGLA). The bulk of this newly formed DGLA is incorporated into neutral lipids and specifically triacylglycerides. Neutrophils from volunteers supplemented with GLA as borage oil also had elevated quantities of DGLA but not GLA, when compared with neutrophils from volunteers not consuming the GLA supplement. To determine whether DGLA could be mobilized from cellular glycerolipids, neutrophils were stimulated with ionophore A23187 and fatty acid levels were determined. DGLA and AA were both released during stimulation, and the quantities of DGLA mobilized increased threefold after in vitro GLA supplementation. Exogenously provided DGLA was converted to one major metabolite during cell stimulation; this product migrated on reverse-phase HPLC with the 15-lipoxygenase product, 15-hydroxy-eicosa-trienoic acid (15-HETre). Both 15-HETre and DGLA (provided exogenously) inhibited the formation of leukotriene B4, (LTB4) and 20-hydroxy-leukotriene B4 (20-OH-LTB4). The IC50 for 15-HETre inhibition of both LTR, and 20-OH-LTB4 in A23187-stimulated neutrophils was 5 microM. This inhibition could be reversed by removing the compounds from the cells. Taken together, these data reveal that there are enzymes within the human neutrophil that metabolize GLA or its elongation product DGLA, and that the metabolism of GLA and AA may interact at a number of critical junctures.
A common TCR beta-chain expressed by CD8+ intestinal mucosa T cells in ulcerative colitis.
Authors: Chott A , Probert C S , Gross G G , Blumberg R S , Balk S P
Published: December 2022

The human intestine contains two populations of anatomically distinct T cells, intraepithelial lymphocytes and lamina propria lymphocytes (LPLs), both of which preferentially use the TCR-alpha beta. Recent studies of TCR alpha- and beta-chain usage by intestinal intraepithelial lymphocytes, which are predominantly CD8+ T cells, have demonstrated that these cells are oligoclonal in normal intestine. This report examined the TCR beta-chains expressed by purified CD4+ and CD8+ T cells from normal colonic lamina propria and from the intestinal mucosa of patients with active ulcerative colitis (UC). The selective expansion of CD8+ T cell clones, and to a lesser extent CD4+ T cell clones, was observed among both normal LPLs and mucosal T cells in UC. These expanded LPL clones from normal donors were all distinct, but the mucosal T cells isolated from five of nine patients with UC contained CD8+ T cells expressing related V beta 3-J beta 1.6 TCRs. These observations provide evidence for an Ag-specific mucosal T cell response in UC. Further studies will be required to identify this Ag and address whether the T cell response to it plays a primary role in initiating the disease or is secondary to the inflammatory response.
Dendritic cells produce macrophage inflammatory protein-1 gamma, a new member of the CC chemokine family.
Authors: Mohamadzadeh M , Poltorak A N , Bergstressor P R , Beutler B , Takashima A
Published: December 2022

Langerhans cells (LC) are skin-specific members of the dendritic cell (DC) family. DC are unique among APC for their capacity to activate immunologically naive T cells, but little is known about their chemotactic recruitment of T cells. We now report that LC produce macrophage inflammatory protein-1 gamma (MIP-1 gamma), a newly identified CC chemokine. MIP-1 gamma mRNA was detected in epidermal cells freshly procured from BALB/c mice, and depletion of I-A+ epidermal cells (i.e., LC) abrogated that expression. MIP-1 gamma mRNA was detected in the XS52 LC-like DC line as well as by 4F7+ splenic DC and granulocyte-macrophage CSF-propagated bone marrow DC. XS52 DC culture supernatants contained 9 and 10.5 kDa immunoreactivities with anti-MIP-1 gamma Abs. We observed in Boyden chamber assays that 1) XS52 DC supernatant (added to the lower chambers) induced significant migration by splenic T cells; 2) this migration was blocked by the addition of anti-MIP-1 gamma in the lower chambers or by rMIP-1 gamma in the upper chambers; and 3) comparable migration occurred in both CD4+ and CD8+ T cells and in both activated and nonactivated T cells. We conclude that mouse DC (including LC) have the capacity to elaborate the novel CC chemokine MIP-1 gamma, suggesting the active participation of DC in recruiting T cells before activation.
A role for c-myc in the regulation of thymocyte differentiation and possibly positive selection.
Authors: Broussard-Diehl C , Bauer S R , Scheuermann R H
Published: December 2022

The purpose of thymocyte differentiation is to establish the T cell repertoire and eliminate nonfunctional and autoreactive T cells. In an analysis of potential regulators of this process, we have found that c-myc expression is down-regulated dramatically during early thymocyte differentiation and subsequently up-regulated along with TCR/CD3 in CD4+8+ cells. Transgenic E beta-myc mice that constitutively express c-myc in thymocytes have a larger proportion of thymocytes with high TCR/CD3 and low heat-stable antigen-1 expression than controls, and an increase in the number of transitional cells with a CD4+CD8low phenotype. Mature E beta-myc T cells respond less vigorously than controls to activation through their TCR/CD3 complex, as measured by proliferation and induction of the activation marker CD69. These results are consistent with a hypothesis in which activation of immature T cells through TCR/CD3 induces c-myc up-regulation and drives thymocytes through the initial stage of positive selection.
Expression and characterization of functionally active recombinant perforin produced in insect cells.
Authors: Liu C C , Persechini P M , Young J D
Published: December 2022

A key cytolytic mediator used by killer lymphocytes, perforin (also known as pore-forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. To overcome the difficulty in obtaining sufficient amounts of perforin and to further assess the functional relevance of the N-terminal portion of the perforin molecule in its lytic activity, we have attempted in the present study to produce recombinant perforins. Three forms of recombinant mouse perforin, a full-length form and two N-terminal truncated forms, have been expressed in insect (Spodoptera frugiperda (Sf9)) cells using recombinant baculovirus. Biochemical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing Ca2+ influx into target cells, and forming structural pores in target membranes. Significant lytic activities were also detected for the two truncated recombinant perforins lacking, respectively, the N-terminal 21 amino acid residues and 121 amino acid residues. Time course study showed that the latter acted less efficiently than the former. These results suggest the N-terminal portion of the perforin molecule to be an important, but not the only, domain responsible for the lytic function of the perforin molecule.
A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis.
Authors: Takabayashi T , Vannier E , Clark B D , Margolis N H , Dinarello C A , Burke J F , Gelfand J A
Published: December 2022

The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF-alpha and IL-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and IL-1 beta. C3a modulated LPS-induced TNF-alpha and IL-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (20-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and IL-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and IL-1 beta. In contrast, in adherent PBMC, C3a at 5 to 20 microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and IL-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and IL-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and IL-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells.
Protective antibodies in murine Lyme disease arise independently of CD40 ligand.
Authors: Fikrig E , Barthold S W , Chen M , Grewal I S , Craft J , Flavell R A
Published: December 2022

Borrelia burgdorferi-infected mice develop acute arthritis that undergoes Ab-mediated resolution. To further investigate the role of B. burgdorferi-specific Abs in Lyme borreliosis, CD40 ligand-deficient (CD40L-deficient) mice were infected with B. burgdorferi. The development and regression of arthritis were similar in CD40L-deficient and control mice. Although CD40L-deficient mice have defects in Ig class switching, infected CD40L-deficient mice developed B. burgdorferi-specific IgG2b Abs. Moreover, the transfer of serum from B. burgdorferi-infected CD40L-deficient animals prevented infection in severe combined immunodeficient mice. These data show that B. burgdorferi-infected CD40L-deficient mice are capable of producing Abs that are protective, despite the inability of these mice to mediate T-dependent immune responses.