bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for an antigen-specific cellular immune response in skin lesions of patients with psoriasis vulgaris.
Authors: Menssen A , Trommler P , Vollmer S , Schendel D , Albert E , Gürtler L , Riethmüller G , Prinz J C
Published: December 2022

Psoriasis vulgaris is an inflammatory skin disease characterized by excessively increased keratinocyte proliferation. Several lines of evidence support the idea that T cells infiltrating psoriatic skin lesions play a vital role in the pathogenesis of the disease. To establish whether lesional accumulation and activation of T lymphocytes reflect a specific local immune response, the TCR beta-chain variable (V beta) region gene usage was studied in chronic psoriatic plaques, normal skin, and paired blood lymphocytes. By semiquantitative PCR, we found that overexpression of either or both V beta 2 and V beta 6 gene families characterized the TCR repertoires of normal skin and psoriatic skin lesions. However, sequence analysis of the complementarity-determining region 3 (CDR3) of these V beta gene families demonstrated a marked TCR oligoclonality only in psoriatic lesions, not in normal skin or in blood lymphocytes. The amino acid sequences of the lesional TCR clones revealed that certain conserved junctional motifs were shared by different patients. A second biopsy taken from one of the psoriasis patients 18 mo later from a different anatomical site disclosed that the same TCR clones were again dominating. These data suggest that lesional psoriatic T lymphocytes expressing the prevailing TCR V beta genes represent an oligoclonal T cell subset that expanded from a few progenitor T cells in response to Ag in the skin of psoriasis patients. They are derived from a polyclonal T cell population that, by the expression of V beta 2 or V beta 6 TCR, appears to be predisposed for homing to the skin.
Paradoxical effects of IL-10 in endotoxin-induced uveitis.
Authors: Rosenbaum J T , Angell E
Published: December 2022

Uveitis, or intraocular inflammation, can be provoked in laboratory rodents by the local or systemic injection of bacterial endotoxin. Many of the inflammatory effects of endotoxin are potentially due to the induction of cytokine synthesis. IL-10 is a cytokine that potently inhibits the synthesis of many cytokines, including IL-1 and TNF-alpha. We have assessed the ability of IL-10 to inhibit endotoxin-induced uveitis in rabbits and mice. The intravitreal injection of 1 micrograms of human recombinant IL-10 was extremely effective in rabbits in reducing the inflammation produced by the intravitreal injection of 250 ng of Escherichia coli endotoxin, as judged by the reduced accumulation of cells and protein in the aqueous humor. Locally injected IL-10 was similarly effective in blocking the ocular inflammatory effects of intravitreally injected endotoxin in a mouse model. If the injection of IL-10 was delayed subsequent to the endotoxin injection, the reduced inflammatory effects in the rabbit model were diminished. In contrast to its ability to inhibit the local inflammatory effect of endotoxin in the eye, IL-10 did not reduce the inflammation induced by a local ocular injection of 400 U of human recombinant IL-alpha. Paradoxically, in a mouse model of uveitis subsequent to intraperitoneally injected endotoxin, the simultaneous injection of 1 micrograms of IL-10 and endotoxin potentiated the ocular inflammation, as judged by the number of leukocytes seen in histologic sections. This effect was dose dependent, since eye inflammation was markedly inhibited by 100 micrograms of IL-10 injected i.p. These observations are compatible with the hypothesis that locally injected IL-10 acts by reducing cytokine synthesis in these uveitis models. Intraperitoneally injected IL-10 can either inhibit or suppress endotoxin-induced eye inflammation in a dose-dependent manner.
Fibroblasts support outgrowth of splenocytes simultaneously expressing B lymphocyte and macrophage characteristics.
Authors: Borrello M A , Phipps R P
Published: December 2022

B lymphocytes and macrophages are considered to be derived from separate lineages and to have specialized functions. However, some malignant B lymphocytes can generate descendants with macrophage-like properties and monocytoid B cells are described in certain diseases. In this report, we demonstrate that normal biphenotypic cells can be isolated by incubating mouse splenic fibroblasts or their conditioned media with purified splenic B lymphocytes. Phagocytic vascular cells were isolated that simultaneously displayed typical B cell (B220, surface IgM, surface IgD, and CD5) and macrophage (F4/80 and Mac-1) markers and contained rearranged Ig genes. F(ab')2 anti-mu Ab inhibited the proliferation of these biphenotypic cells in a dose-dependent manner, indicating that functional IgM was expressed. The adherent B/macrophage cells also displayed CD40 and BCL-2 and were sensitive to ionizing radiation, consistent with having a B cell origin. In the presence of splenic fibroblast-conditioned medium, lines of B/macrophage cells can be propagated for months in vitro. The ability to derive these biphenotypic cells from normal sources suggests that certain B lymphocytes and macrophages share a closer lineage relationship than is predicted by current models of hematopoietic differentiation. Alternatively, these biphenotypic cells may represent a primitive B lymphocyte lineage that possesses greater flexibility to adapt to infectious agents than dedicated lymphoid or myeloid cells and may be prone to malignant transformation.
Identification of a candidate CD5 homologue in the amphibian Xenopus laevis.
Authors: Jürgens J B , Gartland L A , Du Pasquier L , Horton J D , Göbel T W , Cooper M D
Published: December 2022

We identified a novel T cell Ag in the South African clawed toad (Xenopus laevis) by a mAb designated 2B1. This Ag is present in relatively high levels on most thymocytes, approximately 65% of splenocytes, 55% of PBL, and 65% of intestinal lymphocytes, but is rarely seen on IgM+ B cells in any of these tissues. Lymphocytes bearing the 2B1 Ag proliferate in response to stimulation with Con A or PHA, whereas the 2B1- lymphocytes are reactive to LPS. Biochemical analysis indicates that this Ag is a differentially phosphorylated glycoprotein of 71 to 82 kDa. The protein core of 64 kDa bears both N- and O-linked carbohydrate side chains. The amino-terminal protein sequence of the 2B1 Ag shares significant homology with both the macrophage scavenger receptor type 1 motif and the mammalian CD5/CD6 family. The biochemical characteristics and cellular distribution of the 2B1 Ag suggest that it represents the CD5 homologue in X. laevis. While T cells constitutively express this highly conserved molecule, Xenopus B cells acquire the CD5 homologue only when they are stimulated in the presence of T cells.
Comparison of p56lck and p59fyn protein expression in thymocyte subsets, peripheral T cells, NK cells, and lymphoid cell lines.
Authors: Olszowy M W , Leuchtmann P L , Veillette A , Shaw A S
Published: December 2022

The expression of the src-family kinases, p56lck and p59fyn, is critical for thymocyte development and TCR-mediated signal transduction, and may be important for signaling through other lymphoid receptors as well. Overexpression studies have demonstrated that the levels of p56lck and p59fyn expression can affect T cell development and signaling through the TCR. Therefore, it is likely that their exact expression levels play an important role in modulating signaling in thymocytes, mature T cells, and other lymphocytes. Here, we used quantitative immunoblotting to measure p56lck and p59fyn protein expression levels in thymocyte subsets, peripheral T cells, NK cells, and lymphoid cell lines. p59fyn expression levels were similar to p56lck in most cells that were examined demonstrating that p59fyn is abundantly expressed in T cells. In addition, we found that p56lck protein expression is equivalent in CD4 and CD8 double-negative, double-positive, and single-positive thymocytes. In contrast, p59fyn expression levels were significantly lower in double-positive thymocytes than in the other thymocyte subpopulations. Finally, we demonstrate that p56lck and p59fyn expression varies greatly in a number of cell lines used to study T cell activation and that IL-2 treatment can dynamically regulate p56lck and p59fyn expression in some cells.
Functional expression of CD40 antigen on human epidermal Langerhans cells.
Authors: Péguet-Navarro J , Dalbiez-Gauthier C , Rattis F M , Van Kooten C , Banchereau J , Schmitt D
Published: December 2022

It is now well established that interactions of CD40 on the B cells, along with its ligand (CD40-L) on the T cells, regulate B cell proliferation and differentiation. However, the functional significance of CD40 expression on cells known for most efficient Ag-presenting function, i.e., dendritic cells, is not so clear. In this study, we demonstrate that CD40 is expressed on human dendritic Langerhans cells (LC) freshly isolated from epidermis. Using CD40-L transfected cells, CD40 triggering was found to enhance LC viability when cultured and to result in phenotypic alterations. Thus, a 2-day CD40 activation induced up-regulation of CD54 and CD86 at the LC surface, while it did not significantly affect the levels of HLA-DR, CD1a, CD58, and CD80 expression. These phenotypic changes correlate with enhanced LC allostimulatory property, as shown by the use of paraformaldehyde-fixed LC. Furthermore, mAbs against CD40, as well as CD40-L, strongly inhibit the primary T cell response to allogeneic LC. Collectively, these data support a role for CD40/CD40-L pair in the development of normal T cell functions.
Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules.
Authors: Kondo A , Sidney J , Southwood S , del Guercio M F , Appella E , Sakamoto H , Celis E , Grey H M , Chesnut R W , Kubo R T , Sette A
Published: December 2022

The binding capacity of large sets of peptides corresponding to naturally occurring sequences and carrying previously defined A24-specific motifs was analyzed. It was found that only a minority (9-25%) of the motif-carrying peptides bound the relevant HLA-A molecule with good affinity (IC 50% < or = 50 nM), while the majority of them bound only weakly or not at all (IC 50% > or = 500 nM). By correlating the presence of specific residue types at each position along the peptide sequence with average binding affinity, the prominent influence of specific secondary interactions (secondary anchor residues) was revealed. Moreover, secondary interactions appeared to be size-dependent in that the specific effects detected differed in 9-mer and 10-mer peptide sets. Based on these observations, A24-specific refined motifs were also established for both 9-mer and 10-mer ligands, and their merit was verified by testing the binding capacity of independent sets of synthetic peptides. Such refined motifs should facilitate accurate prediction of potential A24-restricted peptide epitopes. It was also noted that certain crucial secondary interactions appear to be remarkably similar in the case of A24 and other HLA-A molecules previously analyzed (A*0201, A3, A11, and others). This may reflect contributions to binding affinity of relatively invariant residues located within the polymorphic pockets of the HLA binding groove.
Mechanisms of T cell epitope immunodominance analyzed in murine listeriosis.
Authors: Safley S A , Jensen P E , Reay P A , Ziegler H K
Published: December 2022

We demonstrate conclusively that the bacterial exotoxin listeriolysin O (LLO) is a target Ag for eliciting CD4+ T cell responses following infection with Listeria monocytogenes. The minimal I-Ek-restricted immunodominant CD4+ T cell epitope was identified as peptide 215-226 (p215-226). Most LLO-specific T cell hybridomas recognized p203-226, p208-226, p215-226, and p215-234, although each exhibited a characteristic pattern of preferential reactivity. One hybridoma (IIIC5) reacted to p203-226 but not to p208-226 or any other LLO peptide tested. With APCs from B10 congenic mice and cells transfected with either I-Ak or I-Ek, IIIC5 recognized p203-216 with I-Ak, while a different hybridoma (IB5) recognized p215-226 with I-Ek. Competitive binding studies demonstrated that of 15 LLO peptides tested, only p203-226, p215-226, and p215-234 had high affinity for I-Ek, while p203-226 could also bind to isolated I-Ak. Of nine LLO peptides tested, only p215-234 bound multiple class II MHC alleles. These findings suggest that the immunodominance of p203-226 may be due in part to the presence of multiple T cell epitopes with I-Ek- and I-Ak-binding capability. Many of the rules of immunodominance observed with model Ags are also operative in our murine model of bacterial infectious disease. Furthermore, a novel mechanism of immunodominance based on newly defined structural features of MHC molecules is implicated. This information is crucial for rational vaccine development.
Experimental Listeria meningoencephalitis. Macrophage inflammatory protein-1 alpha and -2 are produced intrathecally and mediate chemotactic activity in cerebrospinal fluid of infected mice.
Authors: Seebach J , Bartholdi D , Frei K , Spanaus K S , Ferrero E , Widmer U , Isenmann S , Strieter R M , Schwab M , Pfister H , Fontana A
Published: December 2022

In bacterial meningitis, the recruitment of leukocytes across the blood-brain barrier into the central nervous system may be crucial for both elimination of pathogens and tissue injury. In addition to bacterial cell wall products, host factors including chemokines may lead to accumulation of phagocytes within the central nervous system. As shown by Northern analysis, brains of mice infected intracerebrally with Listeria monocytogenes (LM) express mRNA for three chemokines, the macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and MIP-2. The cellular sources of these chemokines comprise both the blood-derived polymorphonuclear leukocytes (PMNs) and monocytes infiltrating the meninges, the ventricular system and the periventricular area. In the course of meningitis a time-dependent increase of MIP-1 alpha and MIP-2 was found in the cerebrospinal fluid (CSF) by ELISA. CSF taken 24 h after infection (CSF-LM24) induced migration of human leukocytes when treated in chemotactic chambers in vitro. Neutralizing Abs to chemokines identified MIP-1 alpha and MIP-2 to be responsible for CSF-LM24 mediated chemotaxis of monocytes and PMNs, respectively. CSF obtained from mock-infected animals contained no MIP-1 alpha or MIP-2 and did not lead to migration of leukocytes. When testing CSF-LM24 on mouse spleen cells, the chemotactic activity detected for mononuclear cells was only partly inhibited by Abs to MIP-1 alpha and -1 beta. Thus, in addition to MIP-1 and -2 other not yet defined chemotactic factors are of importance for recruitment of leukocytes in bacterial meningitis.
Fc gamma II receptor-mediated platelet activation induced by anti-CD9 monoclonal antibody opens Ca2+ channels which are distinct from those associated with Ca2+ store depletion.
Authors: Kuroda K , Ozaki Y , Qi R , Asazuma N , Yatomi Y , Satoh K , Nomura S , Suzuki M , Kume S
Published: December 2022

Anti-human platelet CD9 mAb, NNKY1-19, induced platelet activation in a Fc gamma RII-dependent manner in terms of aggregation and secretion of intracellular granule contents. These responses were considerably suppressed by aspirin. [Ca2+]i elevation in the absence of extracellular Ca2+ ([Ca2+]e), which represents the amount of Ca2+ released from intracellular Ca2+ ([Ca2+]i) stores, was also greatly reduced, whereas Ca2+ influx was sustained at similar levels. We thus investigated the mechanism that leads to the opening of Ca2+ channels in platelets incubated with aspirin. IP3 production and Ca2+ efflux were below detectable levels. 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading of platelets to chelate [Ca2+]i did not reduce Ca2+ influx, as assessed by 45Ca2+ measurement. These findings suggested that NNKY1-19 induces Ca2+ channels to open without [Ca2+]i mobilization or by depleting the [Ca2+]i stores. The magnitude of Ca2+ influx was evaluated by adding [Ca2+]e to a platelet suspension activated by various agonists in the absence of [Ca2+]e. The dose dependence of the Ca2+ influx on [Ca2+]e concentrations differed according to the mode of activation. The ED50 value of Ca2+ after thrombin or thapsigargin stimulation was 0.6 mM, whereas that of NNKY1-19 activation was about 3 mM. The addition of anti-Fc gamma RII mAb, IV.3, even 10 min after the initiation of platelet activation induced by NNKY1-19, inhibited the Ca2+ influx. These findings suggest that the Fc gamma RII-dependent activation of platelets induced by NNKY1-19 directly opens Ca2+ channels, which are distinct from those opened by thrombin or thapsigargin.
Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor.
Authors: Pazdrak K , Justement L , Alam R
Published: December 2022

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.
Rat neutrophil defensins. Precursor structures and expression during neutrophilic myelopoiesis.
Authors: Yount N Y , Wang M S , Yuan J , Banaiee N , Ouellette A J , Selsted M E
Published: December 2022

Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.
Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts.
Authors: Monteiro J , Batliwalla F , Ostrer H , Gregersen P K
Published: December 2022

Long term in vitro culture of clonally expanded CD8+T cells, generally found within the CD57+ or CD28-subset, has generally been unsuccessful, suggesting that these cells may have a limited replicative potential. Telomeric shortening may reflect the action of a "mitotic clock" regulating the number of divisions a cell can undergo. In this study, we have compared the telomeric lengths of CD28-CD8+ and CD28+CD8+ T cells in 10 normal individuals to assess their replicative history. Overall, the telomeric lengths were found to be significantly shorter in the CD28-CD8+ T cell subset compared with the CD28+CD8+ subset. Furthermore, clonally expanded TCRBV11+CD8+ T cells from an individual exhibited telomeric lengths that were 2.9 kb shorter than those found in the polyclonal CD28+CD8+ T cell subset. These findings indicate that clonally expanded CD28-CD8+ T cells have undergone many more rounds of replication than CD28+CD8+ T cells, and consistent with the loss of CD28 expression, they may have reached a state of replicative senescence.
Intercellular adhesion molecule-1 is necessary but not sufficient to activate CD4+ T cells. Discovery of a novel costimulator on kidney tubule cells.
Authors: Hagerty D T
Published: December 2022

Kidney tubule cells (KTC) are targets of T lymphocyte injury during allograft rejection and interstitial nephritis. KTC process and present self- and foreign Ags for immune recognition by CD4+ T cells in vivo and in vitro. However, it is not known whether KTC can provide the costimulatory signal required to fully activate CD4+ T cells. Using the MRL/MpJ fas model of lupus interstitial nephritis, we found that KTC did not express the costimulators B7-1 or B7-2. Nevertheless, KTC from both normal and systemically infected mice provided non-B7 costimulation to splenic CD4+ T cells. T cell proliferation was blocked by mAbs binding intercellular adhesion molecule-1 (ICAM-1) but not by mAb or fusion proteins binding B7-1, B7-2, heat-stable Ag, or vascular cell adhesion molecule-1. Importantly, ICAM-1 expression was necessary but not sufficient to provide costimulation. The transformed KTC line D3.B7- expressed high levels of ICAM-1 but did not provide costimulation. Interestingly, KTC provided costimulation to splenic T cells but not to a Th1 clone. These results show that freshly isolated KTC can provide non-B7 costimulation to splenic T cells via an unidentified costimulator and ICAM-1. Furthermore, these experiments demonstrate the complex nature of T cell activation and show that at least for splenic T cells, three or more signals may be required for full activation on live APC.
Differential activity of dexamethasone on IL-2-, IL-4-, or IL-9-induced proliferation of murine factor-dependent T cell lines.
Authors: Louahed J , Renauld J C , Demoulin J B , Baughman G , Bourgeois S , Sugamura K , Van Snick J
Published: December 2022

Mouse helper T cell lines were developed that proliferate permanently without Ag and APCs in response to either IL-2, IL-4, or IL-9, three cytokines whose receptors interact with the IL-2R gamma-chain for signal transduction. Depending on the growth factor, a marked difference was observed regarding the ability of dexamethasone (DEX) to inhibit cell proliferation. In three different cell lines, proliferation induced by IL-2 was completely arrested, while that supported by IL-9 was hardly affected. With IL-4, proliferation was also maintained but less markedly than with IL-9. Although DEX was able to induce apoptosis in these cells, the inhibition of IL-2-induced proliferation was not the result of apoptosis, as this process was equally antagonized by all three factors. Moreover, addition of IL-4 or IL-9 to cultures previously incubated with IL-2 and DEX for several days restored cell proliferation. Finally, autonomous cell variants derived from the factor-dependent cell lines were still protected by IL-4 and IL-9 against growth inhibition by DEX. Together, these results indicate that growth stimulation in the presence of glucocorticoids and inhibition of apoptosis involve distinct aspects of cytokine activities.
T cell tolerance is influenced by concomitant T cell recognition of cross-reactive self-peptides.
Authors: Tam R C , Fedoseyeva E V , Moskalenko M , Garovoy M R , Benichou G
Published: December 2022

Although the current dogma of T cell recognition stresses its exquisite specificity, T cell clones selected for a given peptide can recognize other sequentially or structurally related peptides. Here, we have examined the immunogenicity and tolerogenicity of various self-peptides derived from region 61-80 of different MHC class I proteins co-expressed in the same mouse. Following immunization of B10.A mice (K(k), A(k), E(k), L(d), D(d)) with self-L(d) 61-80 peptide, vigorous MHC class II-restricted T cell proliferation was elicited after restimulation with either the immunogen or with self-K(k) 61-80 but not with self-D(d) 61-80. Furthermore, adult B10.A mice, tolerized with L(d) 61-80 prior to immunization with L(d) 61-80 did not respond to challenge with L(d) 61-80 and the cross-reactive K(k) 61-80. However, following K(k) 61-80 immunization, L(d) 61-80-tolerized mice responded to K(k) 61-80 but not to L(d) 61-80. Thus, tolerance induction to L(d) 61-80 resulted in the elimination/inactivation of L(d) 61-80-reactive T cells including the subpopulation that cross-reacted with K(k) 61-80. However, T cells that recognized K(k) 61-80 exclusively were preserved. Moreover, we showed that immunization with K(k) 61-80 resulted in tolerance breakdown to the cross-reactive, dominant self-peptide D(b) 61-80 in B10.A(4R) mice (K(k), A(k), L(d),D(b)). Together, these results show that the autoimmune T cell repertoire is influenced by the concomitant recognition of different cross-reactive self-peptides within the same individual.
Regulation of lymphocyte homing into the brain during viral encephalitis at various stages of infection.
Authors: Irani D N , Griffin D E
Published: December 2022

The passage of circulating lymphocytes into the central nervous system (CNS) during acute viral encephalitis was studied in vivo using fluorescently labeled cells inoculated into Sindbis virus (SV)-infected mice. Donor lymphocytes were detected in the brains of recipient animals when mononuclear cells were isolated from the CNS and screened by flow cytometry. The magnitude of this accumulation related to the duration of encephalitis in recipient mice and to the activation state of the inoculated cells. While Ag specificity did not influence lymphocyte entry into the inflamed CNS at any stage of infection, SV-immune cells were retained selectively within the brains of infected animals compared with cells of an irrelevant specificity. Coincident with the onset of CNS inflammation, ICAM-1 and VCAM-1 were up-regulated on cerebrovascular endothelium. Lymphocyte entry into the brains of infected animals during maximal inflammation could be inhibited by pretreating inoculated cells with Abs that blocked LFA-1, but not with those that blocked VLA-4 or down-regulated CD44. None of these reagents prevented lymphocyte entry into the brain at the onset of inflammation, suggesting that the earliest recruited cells utilize presently uncharacterized receptor-ligand interactions. These data show that the degree of existing inflammation and the activation state of circulating cells, but not their Ag specificity, influence lymphocyte recruitment into the brain during SV encephalitis. While CNS homing can be blocked with Abs against known adhesion molecules during peak inflammation, lymphocyte entry into the brain during early infection remains poorly characterized.
Activation of human monocytes through CD40 induces matrix metalloproteinases.
Authors: Malik N , Greenfield B W , Wahl A F , Kiener P A
Published: December 2022

The activation of monocytes/macrophages to secrete pro-inflammatory cytokines and matrix metalloproteinases (MMPs) is critically important in the development of chronic inflammatory diseases. However, the consequence of interactions between activated T cells and monocytes in these inflammatory processes is not well understood. In this study we have investigated the induction of MMPs in human monocytes by activated T cells. We show that fixed cells and the cell membranes from a T cell line, BMS-2, that expresses high levels of the CD40 ligand gp39 (also called TRAP, TBAM, or CD40L) stimulate both the expression of mRNA and the production of MMPs by human monocytic cells. Activation of monocytes by the human T cells could be significantly inhibited by a F(ab')2 fragment of a neutralizing Ab specific for human gp39, but not by an Ab that recognizes murine gp39. Furthermore, recombinant soluble gp39 (sgp39) alone induced marked increases in the levels of a 92-kDa metalloproteinase (gelatinase) in both the human monocytic cell line, THP-1, and peripheral human monocytes, and induction was blocked by the anti-human gp39 Ab. Pretreatment with IFN-gamma significantly enhanced gp39 induction of MMPs in THP-1 cells but not in peripheral monocytes. Up-regulation of mRNA for the 92-kDa MMP by gp39 could be detected within 6 h of stimulation and was maximal 24 h after treatment. MMP enzymatic activity was detectable in the culture medium 12 to 18 h following stimulation of the cells and remained high through 48 h. These results suggest the interaction of T cells with monocytes/macrophages via the gp39-CD40 counter receptors may be significant in development or maintenance of chronic inflammatory lesions.
TCR-mediated death of mature T lymphocytes occurs in the absence of p53.
Authors: Boehme S A , Lenardo M J
Published: December 2022

The p53 protein plays an important role in various forms of thymocyte apoptosis, however its role in mature T lymphocyte death caused by TCR stimulation has not been examined. We demonstrate here that T cell blasts derived from mice containing a germ-line deficiency of the p53 tumor suppressor gene are susceptible to TCR-induced apoptosis to the same degree as wild-type T cells. TCR stimulation of both resting and proliferatingT cells results in up-regulated expression of the p53-induced genes Bax and p21, and the induction of these genes appears reduced in T cells that are deficient in p53. Thus, while activation of p53-dependent genes following TCR stimulation is defective in p53-/- T cells, there is no impairment in their ability to undergo apoptosis. These results suggest that TCR-mediated apoptosis of mature T cells takes place via a pathway that is independent of p53.
Epicutaneous exposure of protein antigen induces a predominant Th2-like response with high IgE production in mice.
Authors: Wang L F , Lin J Y , Hsieh K H , Lin R H
Published: December 2022

Hapten-induced contact hypersensitivity has been well-characterized in humans as well as in animal models. However, it is not clear whether or not protein Ag can directly sensitize epicutaneously and induce a primary immune response. We demonstrated in this study, for the first time, that through epicutaneous exposure protein Ag in the absence of adjuvant sensitizes animals and induces a predominant Th2-like response. Furthermore, mice receiving repeated protein Ag sustained elevated levels of specific IgE. This animal model can be used to investigate the molecular mechanism controlling the differential Th1/Th2 development in skin diseases.
Intracellular signaling pathways involved in the induction of apoptosis in immature thymic T lymphocytes.
Authors: Anderson K L , Anderson G , Michell R H , Jenkinson E J , Owen J J
Published: December 2022

We describe a novel technique for studying the signaling pathways that control thymocyte negative selection which maintains the essential interactions between thymocytes and thymic stromal cells. Bisected lobes from newborn mouse thymus are maintained in organ culture for up to 36 h, and the thymocytes analyzed by flow cytometry. Inclusion of [3H]inositol during culture allows measurements of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) hydrolysis and inositol phosphate accumulation. Using this technique we have compared the thymocyte responses induced by anti-CD3, anti-Fas, Con A, and beta-adrenergic stimulation. We show that PtdIns(4,5)P2 hydrolysis precedes anti-CD3-induced thymocyte apoptosis, but not the apoptosis induced by anti-Fas. In contrast, Con A stimulates PtdIns(4,5)P2 hydrolysis, but does not induce thymocyte apoptosis. Anti-CD3, anti-Fas, and Con A all fail to change thymic cAMP levels, but beta-adrenergic stimulation causes a large increase in intracellular cAMP, and agents that elevate cAMP induce thymocyte apoptosis. Inhibition of protein synthesis (with cycloheximide or emetine) prevents the apoptosis induced by anti-CD3 and elevated cAMP, but not that induced by anti-Fas, whereas protease inhibition (with 3,4-dichloroisocoumarin or N(alpha)-tosyl-phenylalanine chloromethyl ketone) prevents the apoptosis caused by all of the effective stimuli. These results offer three important conclusions. First, activation of a variety of different signaling pathways can bring about thymocyte apoptosis. Second, ligation of the thymocyte TCR/CD3 complex provokes PtdIns(4,5)P2 hydrolysis, but signaling through this pathway alone does not necessarily lead to apoptosis. Third, by whichever signaling pathway the response is initiated, the activity of one or more protease enzymes appears to form an essential component in the final common pathway leading to apoptosis.
Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide-38 inhibit IL-10 production in murine T lymphocytes.
Authors: Martinez C , Delgado M , Gomariz R P , Ganea D
Published: December 2022

Vasoactive intestinal peptide (VIP), a neuropeptide present in the peptidergic innervation of lymphoid organs and expressed in thymocytes and peripheral lymphocytes has been previously reported to modulate cytokine expression in T lymphocytes. In this study, we investigated the effects of VIP and of the structurally related neuropeptide PACAP-38 on the expression of IL-10 in murine lymphocyte cultures. Both neuropeptides inhibit IL-10 production by spleen cells or thymocytes activated via the TCR-associated CD3 complex in a similar dose-response manner. The inhibition is specific, presumably mediated through the VIP-R1, and maximum inhibitory levels are achieved within the first 5 to 15 min of exposure to VIP or PACAP-38. CD4+ T cells function as direct cellular targets for the two neuropeptides. The fact that VIP, PACAP-38, and forskolin, all known cAMP inducers, also inhibit IL-10 production, suggests the participation of cAMP in signal transduction. VIP and PACAP-38 regulate transcriptional expression of IL-10, since IL-10 steady state mRNA levels are significantly reduced by treatment with the two neuropeptides. These results expand the range of neuroendocrine-regulated cytokines and support the idea that neuropeptides such as VIP and PACAP, which are released or produced in the local lymphoid microenvironment and specifically modulate the expression of various cytokines, may participate in the intricate cytokine network controlling local immune responses.
Modulation of the expression of the IFN-gamma receptor beta-chain controls responsiveness to IFN-gamma in human peripheral blood T cells.
Authors: Sakatsume M , Finbloom D S
Published: December 2022

IFN-gamma has potent antiproliferative and apoptotic effects in T cells that are important in determining T cell development and polarized differentiation. Therefore, any event that enables T cells to become less responsive to IFN- gamma may potentially alter immune responsiveness to Ag. In this work, we show that human peripheral blood T cells that are stimulated through the TCR and expanded with IL-2 are unresponsive to IFN-gamma, as determined by a lack of activation of jak kinases and the transcription factor, STAT1(alpha), a signal transducer and activator of transcription. This nonresponsiveness occurs because of a lack of expression of the beta- chain (accessory factor) of the IFN-gamma receptor, while at the same time maintaining IFN-gamma receptor alpha-chain expression. Expression of the beta-chain can be restored by secondary TCR ligation or PMA treatment. T cell blasts treated with PMA are now responsive to IFN-gamma. When freshly isolated, highly enriched (>98%) T cells are examined for IFN-gamma responsiveness; these cells can respond to IFN-gamma and express beta-chain. Therefore, as T cells progress from primary TCR activation through IL-2-dependent proliferation, followed by secondary TCR stimulation, their responsiveness to IFN-gamma varies, and this may affect their ability to participate in an ongoing immune response.
A novel substrate-binding pocket interaction restricts the specificity of the human NK cell-specific serine protease, Met-ase-1.
Authors: Smyth M J , O'Connor M D , Trapani J A , Kershaw M H , Brinkworth R I
Published: December 2022

Human Met-ase-1 is a NK cell-specific member of a family of serine proteases (granzymes) that participate in target cell death inflicted by cytotoxic lymphocytes. This granzyme is predicted to cleave to the carboxyl side of long narrow hydrophobic amino acids (such as methionine), but not large, bulky hydrophobic amino acids (such as phenylalanine). To study the key structural features that confer this unusual serine protease specificity, active recombinant human Met-ase-1 was expressed in COS-7 cells. Protease assays of transfected COS-7 cell lysates provided evidence that an activation prohexapeptide normally regulates processing of this granzyme in NK cells. Recombinant human Met-ase-1 cleaved thiobenzylester substrates specifically after methionine, norleucine, or leucine residues in the primary substrate site (P1). Two key residues of human Met-ase-1, Lys179 Met (approximately chymotrypsin CHA192) and Ser201Gly (approximately CHA216), were mutated based upon a model structure derived from the crystal structure of chymotrypsin A. These mutants had reduced activity for substrate containing methionine at P1, but acquired chymase activity for phenylalanine at P1. Lys179 Met and Ser201Gly in the substrate-binding pocket of human Met-ase-1 restrict the preference of this granzyme for long narrow hydrophobic amino acids in the P1. A potential hydrogen-bonding interaction between these two residues on opposing sides of the substrate-binding pocket represents a novel molecular mechanism by which lymphocyte serine proteases might provide greater substrate specificity.
The J chain gene is transcribed during B and T lymphopoiesis in humans.
Authors: Bertrand F E , Billips L G , Gartland G L , Kubagawa H , Schroeder H W
Published: December 2022

In mice and chickens, J chain appears to be expressed only in activated B cells and plasma cells. In humans, studies based mainly on transformed cells suggest that J chain expression may initiate during earlier stages in B lineage differentiation. In the present study, we isolated a series of hematopoietic subpopulations from human fetal and adult tissues by immunofluorescence cell sorting and examined each subpopulation for J chain expression by reverse transcriptase-PCR. In fetal and adult bone marrow, J chain transcripts were detected at all stages of B lineage differentiation, including the progenitor (CD34+/CD19-) and pro-B (CD34+/CD19+) cell subpopulations. J chain mRNA was also detected during fetal thymocyte development: double negative (CD4-/CD8-) through single positive (CD4+ or CD8+) cell subpopulations. The J chain message was not detected in peripheral CD3+ T cells, CD14+ monocytes, and CD56+ NK cells from either fetal or adult samples. The nucleotide sequence of J chain PCR products from CD34+/CD19- bone marrow progenitors and CD4+/CD8- thymocytes proved identical to the previously reported sequence of functionally spliced J chain mRNA. These results suggest that the J chain gene is transcriptionally active during early stages of both B cell and T cell differentiation, before the expression of their respective Ag receptors.
Local intestinal immune responses to infections with Trichinella spiralis. Real-time, continuous assay of cytokines in the intestinal (afferent) and efferent thoracic duct lymph of rats.
Authors: Ramaswamy K , Negrao-Correa D , Bell R
Published: December 2022

Levels of IL-4, IL-5, TNF-alpha, and IFN-gamma were quantitated in the intestinal (afferent) and efferent thoracic duct lymph of rats during the course (0 to 289 h) of an infection with Trichinella spiralis. Intestinal lymph was collected by cannulating thoracic ducts of mesenteric lymphadenectomized animals. These studies showed that cytokines typical of a Th2 type (IL-4 and IL-5) and a Th1 type (IFN-gamma) were simultaneously detected in the intestinal lymph during the first 8 days after infection. Worm expulsion (day 11 to 12) was associated with increased levels of IL-4 and IL-5 in the intestinal lymph. IL-5 levels rose as early as 15 to 20 h and remained elevated throughout the infection. IL-4 activity appeared in intestinal lymph 60 h after infection and reached peak levels during worm expulsion. Despite the predominantly Th2 nature of cytokine response, IFN-gamma levels showed several cycles of high and low production during the course of infection. A comparison of cytokine levels between intestinal and efferent lymph values showed no significant differences in IL-4 or IL-5 levels suggesting no contribution by the mesenteric node to efferent lymph. However, IFN-gamma and TNF-alpha levels were lower in efferent lymph compared with intestinal lymph suggesting mesenteric node consumption. Adoptive transfer experiments showed that protective CD4+CD45RC- cells primed the gut for a more rapid TH2-type response that was faster than in a primary infection. In contrast, adoptive transfer of CD4+CD45RC+ cells primed the gut for a more rapid Th1-type(IFN-gamma) response. These studies demonstrate a novel method for measuring real-time changes in cytokine levels in the gut during the course of an active infection.
Identification of Fc alpha receptor (CD89) isoforms generated by alternative splicing that are differentially expressed between blood monocytes and alveolar macrophages.
Authors: Patry C , Sibille Y , Lehuen A , Monteiro R C
Published: December 2022

One of the hallmarks of mucosal-host defense is the clearance of inhaled Ags by alveolar macrophages (AM) through interactions of IgA Abs and IgA Fc receptors (Fc alpha R). AM constitutively expressed Fc alpha R at lower levels than freshly isolated and in vitro-differentiated monocytes as determined by immunofluorescence using four anti-Fc alpha R mAb. SDS-PAGE analysis of iodinated cell surface proteins revealed that Fc alpha R on AM has an Mr of 50 to 65 kDa, slightly lower than that on monocytes (55-75 kDa). Treatment of AM Fc alpha R by N-glycanase gave rise to a protein core of 28 KDa, smaller than the 32-kDa backbone of blood monocytes. AM Fc alpha R molecules were unaffected by phosphatidylinositol-phospholipase C treatment. Fc alpha R transcripts were analyzed by reverse transcription-PCR using primers in the 5' and 3' regions of a U937 Fc alpha R cDNA. Three transcripts were amplified, cloned, and sequenced from AM and/or monocyte mRNA, the full length Fc alpha R and two alternatively spliced products corresponding to deletions of 66 and 288 nucleotides in the portion coding for the extracellular domain; they were named Fc alpha R a.1, a.2, and a.3, respectively. These PCR products were transcribed and translated in vitro into three proteins (Mr 32, 30, and 22 kDa, respectively), in which the 32- and 30-kDa species were immunoprecipitated by an anti-Fc alpha R mAb. The predicted size of the protein encoded by the Fc alpha R a.2 transcript without the leader peptide is Mr approximately 27,400, a value that is consistent with the Mr of AM Fc alpha R backbone. These results indicate that AM express at their surfaces a protein product of an alternatively spliced Fc alpha R transcript, the Fc alpha R a.2 isoform, that might have physiologic relevance in IgA-mediated host defense at mucosal sites.
Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides.
Authors: Cowdery J S , Chace J H , Yi A K , Krieg A M
Published: December 2022

Microbial products released during bacterial infection induce cytokine-mediated inflammatory responses that can be protective, but excessive release of inflammatory cytokines may promote development of the sepsis syndrome. We examined the ability of bacterial DNA to induce in vivo cytokine release and to potentiate the toxicity of LPS. Intravenous treatment of mice with Escherichia coli (EC) DNA, but not calf thymus (CT) DNA, induced a rapid (within 4 h) dose-dependent increase in serum IFN-gamma and splenic IFN-gamma-forming cells. Over 90% of splenic IFN-gamma-producing cells were identified by surface phenotype as NK cells. Mice also mounted an IFN-gamma response following challenge with 20-base oligonucleotide that contained an internal CG motif (but did not respond to a control oligonucleotide). Treatment of mice with EC DNA followed by a sublethal LPS challenge resulted in a 3-fold increase in the peak serum level of TNF-alpha and a 10-fold increase in the peak level of IL-6 compared with mice that received CT DNA followed by LPS. Mice treated with EC DNA followed by LPS showed 75% mortality, compared with no deaths in mice treated with CT DNA followed by LPS. EC DNA/LPS treatment of mice with disrupted IFN-gamma genes resulted in a 5% mortality while 59% of similarly treated +/+ mice died. Thus, bacterial DNA induces in vivo release of IFN-gamma which, in turn, is associated with an increase in LPS-induced TNF-alpha and IL-6 release, and with increased sensitivity to the toxic effects of LPS.
CD28 is required for germinal center formation.
Authors: Ferguson S E , Han S , Kelsoe G , Thompson C B
Published: December 2022

Previous studies have demonstrated that the T cell costimulatory molecule, CD28, is important in the development of humoral immunity. CD28-deficient mice exhibit defects in isotype switching and are more susceptible to pathogens that depend on an effective Ab response. To determine the basis of these defects, we have examined B cell responses of CD28-deficient mice at the microenvironmental level. Early in a normal T-dependent immune response, small numbers of B cells undergo activation in the T cell-rich zone of secondary lymphoid tissues and then migrate to B cell areas. These migrant B cells found developing germinal centers by proliferative expansion, during which individual cells acquire mutations in their rearranged Ig genes. B cell mutants retaining higher affinities for Ag undergo positive selection in germinal centers, resulting in the establishment of the memory B cell compartment. In the present study, we demonstrate that although potentially Ag-reactive cells within the lymphoid follicle accumulate following antigenic challenge, these cells fail to undergo proliferative expansion to form germinal centers and do not acquire somatic mutations in CD28-deficient animals. Thus, the CD28 activation pathway is required for Ab responses to T-dependent Ags. cell compartment. In the present study, we demonstrate that although potentially Ag-reactive cells within the lymphoid follicle accumulate following antigenic challenge, these cells fail to undergo proliferative expansion to form germinal centers and do not acquire somatic mutations in CD28-deficient animals. Thus, the CD28 activation pathway is required for Ab responses to T-dependent Ags.