bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential regulation of C alpha 1 and C alpha 2 germ-line and mature mRNA transcripts in human peripheral blood B cells.
Authors: Kitani A , Strober W
Published: December 2022

In this study, we investigated the regulation of germ-line and mature C alpha mRNA transcript expression in human peripheral blood B cells. In initial studies, we found that C alpha germ-line and mature transcripts were constitutively expressed in total peripheral blood B cells but not in high density surface IgM+ (surface IgA-) B cells; the latter cells were therefore used in subsequent studies. Focusing first on regulation of germ-line (I alpha-C alpha) transcripts, we showed that C alpha 1 germ-line transcripts are induced by TGF-beta 1 alone but such induction is enhanced by Staphylococcus aureus, Cowan I (SAC). In contrast, C alpha 2 germ-line transcripts are optimally induced by TGF-beta 1 in the absence of a B cell stimulus. In addition, although SAC alone induced both C alpha 1 and C alpha 2 germ-line transcripts, such induction is dependent on endogenous (B cell-derived) TGF-beta 1 production, because no induction is detected in cells cultured in the presence of neutralizing anti-TGF-beta 1. Turning next to mature (VDJ-C alpha) transcripts we showed that SAC plus TGF-beta 1 induces mature C alpha 1 transcripts, and such induction is IL-10 dependent because it is enhanced by exogenous IL-10 and abrogated by the presence of anti-IL-10. In contrast, SAC plus TGF-beta 1 does not induce C alpha 2 mature transcripts, even in the presence of exogenous IL-10; such transcripts are induced, however, by T cell stimuli such as anti-CD40 (presented by CDw32-transfected L cells) in the presence of TGF-beta 1/IL-10 or by PWM-activated T cells. In summary, these studies show that C alpha 1 and C alpha 2 germ-line transcript induction is differentially regulated, in keeping with previous studies of differences in germ-line CH transcript induction in the first and second IgH duplication units. Furthermore, C alpha 1 and C alpha 2 mature transcript induction is also differentially regulated, with C alpha 2 requiring a T cell signal such as that delivered by the CD40 ligand. Finally, IL-10 appears to be uniquely supportive of the induction of both C alpha 1 and C alpha 2 mature transcripts.
Evidence that the activation of an inactive pool of membrane-associated protein kinase C is linked to the IL-2-dependent survival of T lymphocytes.
Authors: Lu Y , Tremblay R , Jouishomme H , Chakravarthy B , Durkin J P
Published: December 2022

The activation of protein kinase C (PKC) is believed to result from the translocation of inactive cytosolic enzyme to the lipid environment of membranes. However, by using a novel method for measuring PKC activity directly in isolated membranes, we have previously shown that a significant proportion of the PKC present in a variety of cells associates with membranes in an inactive state, and that this pool of inactive PKC can be stimulated specifically in cells in the absence of translocation. IL-2 did not stimulate the translocation of PKC to membranes in the IL-2-dependent mouse T cell line, CTLL-2. Nevertheless, a transient, two approximately threefold increase in membrane PKC activity was observed within 10 min of IL-2 addition to these cells. This increase was entirely caused by the activation of a pool of inactive membrane PKC previously associated with the membrane. The inhibition of PKC activity by the specific inhibitors bisindolylmaleimide (BIS) and 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) blocked the ability of IL-2 to suppress the onset of apoptosis in IL-2 and serum-deprived CTLL-2 cells. The inhibition of this important function of IL-2 was most pronounced when the PKC inhibitors were added to the medium within 2 h of stimulating the cells with IL-2. The results suggest that transient activation of inactive membrane PKC is linked to the IL-2 receptor signaling, and may be an important step in the mechanism(s) by which the cytokine suppresses cell death in T lymphocytes.
Characterization of preneoplastic thymocytes and of their neoplastic progression in irradiated C57BL/Ka mice.
Authors: Sen-Majumdar A , Guidos C , Kina T , Lieberman M , Weissman I L
Published: December 2022

Mice that receive whole body split-dose irradiation develop thymic lymphomas after a long latent period. Before emergence of frank lymphomas, preneoplastic thymocytes, which are defined by their ability to progress to full malignancy on intrathymic transfer to congenic hosts, appear. A combination of mAb 1C11, which binds to a cell surface glycoprotein on lymphoma cells, and of Abs to the differentiation markers CD4 and CD8 (MHC co-receptors), and CD3 (TCR complex) was used to characterize the phenotypes of preneoplastic thymocytes and to place them within the scheme of normal T cell ontogeny. In the irradiated, preneoplastic thymus, the 1C11 molecule was found to be present on CD4-8-, CD4-8+, and CD4+8+, but not CD4+8-, cells. After intrathymic transfer to Thy-1 congenic recipients, 1C11highCD4-8- cells from irradiated mice showed the highest leukemogenic potential, followed by the 1C11highCD4-8+ and 1C11highCD4+8+ subsets. Within the 1C11highCD4-8- subset, CD3+ cells were more leukemogenic than CD3- cells. The resulting lymphomas were 1C11highCD3+4-8+ and 1C11highCD3+4+8+, phenotypes that are absent or very rare in the normal thymus, but similar to those of primary radiation-induced lymphomas. Examination of the TCR V beta repertoire in these lymphomas shows a highly significant bias, in that approximately 50% express the V beta 8 gene product. These results indicate, but do not prove, that the 1C11highCD3+4-8- cells, a very small subset of normal thymocytes, are either the target of neoplastic transformation after radiation or the earliest identifiable cell population after the transforming event. These results also suggest at least one possible pathway to define the process leading to overt lymphoma.
Adenosine and homocysteine together enhance TNF-mediated cytotoxicity but do not alter activation of nuclear factor-kappa B in L929 cells.
Authors: Bergmann S , Shatrov V , Ratter F , Schiemann S , Schulze-Osthoff K , Lehmann V
Published: December 2022

This paper shows that a combination of adenosine and homocysteine potentiates TNF-alpha-mediated cytotoxicity, but does not modulate activation of NF-kappa B transcription factor controlling the expression of various TNF-alpha-inducible genes. Adenosine and homocysteine at concentrations (1 mM each) that enhance TNF-alpha-induced cytotoxicity accumulate S-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of S-adenosyl-L-methionine-dependent methylation reactions. In addition, preloading L929 cells with AdoHcy resulted in enhanced responses to TNF-alpha, suggesting that AdoHcy potentiates TNF-alpha-induced cytotoxicity. Moreover, the combination of adenosine and homocysteine changed the dependency of the TNF-alpha-mediated cytolysis on reactive oxygen intermediates. In the absence of adenosine and homocysteine TNF-alpha-mediated cytotoxicity was inhibited by antioxidants such as butylated hydroxyanisole and pyrrolidine dithiocarbamate. In the presence of adenosine and homocysteine, however, TNF-alpha-mediated cytotoxicity is not inhibited by these antioxidants. A L929 subclone, defective in the respiratory chain, resisted the cytotoxic action of TNF-alpha, but was rendered TNF-alpha sensitive in the presence of adenosine and homocysteine. Unlike TNF-alpha-mediated cytotoxicity, the TNF-alpha-induced activation of NF-kappa B was inhibited by antioxidants regardless whether adenosine and homocysteine were present or absent in the culture medium. In conclusion, a combination of adenosine and homocysteine selectively modulates TNF-alpha-mediated cytotoxicity without changing the TNF-alpha-induced activation of NF-kappa B. Our results could facilitate the development of strategies that permit dissection of cytotoxic and gene-activating pathways.
Human T lymphocyte chemotaxis and adhesion induced by vasoactive intestinal peptide.
Authors: Johnston J A , Taub D D , Lloyd A R , Conlon K , Oppenheim J J , Kevlin D J
Published: December 2022

Vasoactive intestinal peptide (VIP), a 28-amino acid peptide of the glucagon-secretin family, has been reported to have significant immunoregulatory properties. In the present study, we demonstrate that VIP has potent chemotactic effects on T lymphocytes. At concentrations of between 10(-8) and 10(-9) M, VIP stimulated significant in vitro chemotaxis of T lymphocytes from both CD4+ and CD8+ subsets. VIP produced more potent chemotactic effects on unstimulated T cells than on anti-CD3-activated cells. Following anti-CD3 activation, binding of 125I-labeled VIP to T cells was reduced and this correlated with a reduction in receptor number without any change in affinity. Preincubation of unstimulated T cells with VIP produced increases in adhesion to ICAM and VCAM integrins. In addition, VIP pretreatment significantly increased unstimulated T cell adhesion to the extracellular matrix protein fibronectin. However, VIP induced less binding of activated T cells to fibronectin. Taken together these results suggest that VIP is a potent chemoattractant and stimulant of adhesion for T lymphocytes. In contrast to chemokines that are more active on stimulated T cells, such as RANTES, this neuropeptide preferentially targets unactivated T cells, suggesting that it may play a greater role in homing and distribution of lymphocytes.
Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.
Authors: Leist M , Gantner F , Bohlinger I , Germann P G , Tiegs G , Wendel A
Published: December 2022

Freshly isolated mouse hepatocytes were essentially insensitive to TNF-alpha cytotoxicity. However, TNF-alpha induced a concentration-dependent cell death in hepatocytes that had been pretreated with the transcriptional inhibitors actinomycin D (ActD), D-galactosamine, or alpha-amanitin. Unlike RNA synthesis inhibition, a translational block in the presence of cycloheximide (CHX) or puromycin did not sensitive hepatocytes to TNF. On the contrary, these agents prevented hepatocytotoxicity induced by ActD/TNF. Pretreatment with peroxides or glutathione depletors had no significant influence on TNF cytotoxicity. In vivo treatment of mice with ActD/TNF caused hepatic failure, which was significantly reduced by co-treatment with CHX. These findings demonstrate that protein synthesis is required for this mechanism of cell death. To test whether TNF may trigger an endogenous suicide program in hepatocytes, we examined whether DNA fragmentation preceded cell death. In the culture system, hepatocellular DNA fragmentation in the presence of ActD/TNF was observed several hours before lactate dehydrogenase release and was inhibited by CHX. Similar results were obtained in vivo. Chromatin condensation and the formation of apoptotic bodies were observed in livers from mice treated with ActD/TNF and significant DNA fragmentation was detected as early as 4 h after challenge. At this time, organ total glutathione content and plasma transaminase levels were not significantly different from those of untreated controls. The findings of this study demonstrate that direct hepatotoxicity of TNF-alpha is associated with an apoptotic mechanism that becomes manifest under the metabolic condition of arrested transcription and functional translation.
Elevation of lymphocyte CD45 protein tyrosine phosphatase activity during mitosis.
Authors: Melkerson-Watson L J , Waldmann M E , Gunter A D , Zaroukian M H , Esselman W J
Published: December 2022

CD45 is an abundant cell-surface protein tyrosine phosphatase (PTPase) of hematopoietic cells that mediates specific tyrosine dephosphorylation in lymphocyte Ag activation. The hypothesis that CD45 PTPase activity varies during progression through the cell cycle was examined in this study. Expression of CD45 PTPase activity was determined in pre-B lymphoma (70Z/3.12) and cytolytic T lymphocyte (CTLL-2) cell lines after fractionation by counterflow centrifugal elutriation into cell cycle-stage-enriched subpopulations. For both cell lines, CD45 PTPase activity was elevated 2- to 10-fold in late G2 + M fractions compared with the PTPase activity of asynchronous cells. FACS and SDS-PAGE analyses indicated that there was only a minor increase in CD45 protein expression during progression through the cell cycle, suggesting that increased CD45 PTPase activity was a result of increased enzymatic activity. Cyclin B expression in 70Z/3.12 cells was evaluated in elutriated subpopulations and intact cyclin B was present in all cell cycle phases from G1 to late G2 + M, disappearing in mitosis and just before those fractions containing maximum CD45 activity. This observation indicated that the peak PTPase activity of CD45 occurred in late mitosis or in cytokinesis. The elevation of CD45 PTPase activity in mitosis supports the hypothesis that CD45 has a role in phosphorylation events occurring during cell division.
Dominant negative mutant of c-Jun inhibits NF-AT transcriptional activity and prevents IL-2 gene transcription.
Authors: Petrak D , Memon S A , Birrer M J , Ashwell J D , Zacharchuk C M
Published: December 2022

Expression of the transcription complex AP-1, composed of Jun and Fos family members, can be induced by a variety of stimuli. In lymphocytes, AP-1 transcriptional activity increases after TCR ligation and plays an important role in T cell activation events such as lymphokine secretion. To explore the requirements for AP-1 in IL-2 production, the AP-1 complex was targeted with a dominant negative mutant c-Jun protein, TAM-67, from which the transactivation domain has been deleted. In transient transfections of Jurkat cells, TAM-67 efficiently inhibited endogenous AP-1 transcriptional activity and blocked the activity of a reporter construct containing the 5' regulatory region of the IL-2 gene. TAM-67 also inhibited the transcriptional activity of nuclear factor-AT (NF-AT), whereas the NF-kappa B, NF-IL-2A, and the proximal TRE-like sites were relatively unaffected. The use of this dominant negative transcription factor suggests that: 1) transactivation-defective nuclear factors represent a novel approach to study the functional consequences of nuclear protein interactions on gene transcription; 2) the proximal TRE-like site from the IL-2 promoter is different from the consensus TRE; and 3) AP-1 plays an important role in the transcriptional activation mediated by the NF-AT binding complex.
IL-4 directly modulates function of a model human intestinal epithelium.
Authors: Colgan S P , Resnick M B , Parkos C A , Delp-Archer C , McGuirk D , Bacarra A E , Weller P F , Madara J L
Published: December 2022

Intestinal epithelia are in intimate contact with subepithelial and intraepithelial lymphocytes. When stimulated, mucosal lymphocytes generate inflammatory cytokines such as IL-4 and IFN-gamma. We have shown that IFN-gamma directly regulates epithelial function. It is unknown whether IL-4 might influence epithelial function and, if so, whether such influences are similar to or differ from those exerted by IFN-gamma. In this study, we examine the effect of human IL-4 on barrier function, ion transport, and immune accessory ligand expression on T84 cells, a crypt-like epithelial cell line. Basolateral exposure of epithelial monolayers to IL-4 attenuated epithelial barrier function by greater than 65% in a dose (50% of effective dose = 1 U/ml)- and time (t1/2 = 24 h)-dependent fashion, and was inhibitable by neutralizing anti-IL-4 and anti-IL-4R Ab. Stimulated Cl- secretion, as measured by epithelial short circuit current, was diminished by as much as 70% by IL-4. Epithelial preexposure to IL-4 brought about a greater than twofold increase in beta 2 integrin-dependent neutrophil adhesion to epithelial, but retarded neutrophil migration into and across epithelial monolayers. ELISAs revealed that epithelial exposure to IL-4 had no effect on cell surface expression of MHC class I, MHC class II, or ICAM-1. These results indicate that IL-4, like IFN-gamma, may serve to regulate intestinal epithelial function, but that resulting phenotypes may be cytokine specific. We speculate from these data that activation of the basolateral receptor for IL-4 potentially provides a new strategy for damping the cellular component of active inflammation in the intestine.
Tenidap modulates cytoplasmic pH and inhibits anion transport in vitro. I. Mechanism and evidence of functional significance.
Authors: McNiff P , Svensson L , Pazoles C J , Gabel C A
Published: December 2022

Tenidap is a novel anti-inflammatory and antiarthritic agent that in clinical studies of rheumatoid arthritis patients, displays symptomatic efficacy superior to nonsteroidal anti-inflammatory drugs (NSAIDs) and equivalent to combinations of NSAIDs and second line agents. Clinical and preclinical biochemical studies have demonstrated that tenidap combines cytokine modulation with suppression of prostaglandin biosynthesis. To better understand tenidap's mechanism of action, in vitro studies of intracellular pH (pHi) were conducted. In cells loaded with the pH-sensitive fluorescence dye 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein, tenidap, but not NSAIDs, caused a rapid and sustained acidification of the cytoplasmic compartment. Tenidap did not act as a proton ionophore, as it did not dissipate the low pH within lysosomes. Mammalian cells regulate pHi through the concerted action of a number of specific transport proteins, including sodium-proton antiporters and chloride-bicarbonate exchangers. Tenidap did not alter pHi via inhibition of the sodium-proton antiporter, but inhibited activity of chloride-bicarbonate exchangers, as did UK5099, a known anion-transport inhibitor that also lowers pHi. This similar activity suggests that the pHi change is coupled to anion transport inhibition. As a result of the pHi change, tenidap affected pH-dependent cellular activities. Tenidap inhibited mannose 6-phosphate receptor-mediated endocytosis, inhibited protein synthesis, and stimulated accumulation of the amino acid leucine. Effects on these cellular processes rapidly reversed when tenidap was removed from the culture medium. Tenidap's in vitro activities were highly dependent on the medium composition; protein content, pH, and bicarbonate concentration all were important factors that influenced activity. These results indicate that tenidap is a potent anion-transport inhibitor and modulator of pHi. Within the appropriate cell or tissue microenvironment, these activities may contribute to tenidap's novel therapeutic profile.
IFN-gamma selectively inhibits lipopolysaccharide-inducible JE/monocyte chemoattractant protein-1 and KC/GRO/melanoma growth-stimulating activity gene expression in mouse peritoneal macrophages.
Authors: Ohmori Y , Hamilton T A
Published: December 2022

IFN-gamma and LPS have both been shown to stimulate enhanced chemoattractant cytokine gene expression in mononuclear phagocytes. In this report, IFN-gamma was found to suppress LPS-induced chemokine mRNA expression in a cell type- and gene-specific fashion. Expression of JE (monocyte chemoattractant protein-1) and KC (GRO/melanoma growth-stimulating activity) mRNA in macrophages stimulated with LPS was markedly suppressed by IFN-gamma in a dose- and time-dependent fashion. LPS-induced IP-10 mRNA was unaffected by IFN-gamma under identical experimental conditions. This effect was cell type-specific because JE and KC mRNA expression in LPS-stimulated murine endothelial cells, TNF-alpha-stimulated endothelial cells, and NIH-3T3 cells were unaffected by IFN-gamma. The IFN-gamma-mediated suppression of LPS-stimulated KC mRNA expression was independent of protein synthesis and mediated at the transcriptional level. These observations indicate that IFN-gamma may function as a negative regulatory signal for the expression of some proinflammatory cytokines in macrophages. The cell type-dependent differential behavior of individual members of the chemokine family may be an important determinant of the cellular composition and outcome of an inflammatory response.
Ligand binding to monocyte alpha 5 beta 1 integrin activates the alpha 2 beta 1 receptor via the alpha 5 subunit cytoplasmic domain and protein kinase C.
Authors: Pacifici R , Roman J , Kimble R , Civitelli R , Brownfield C M , Bizzarri C
Published: December 2022

Regulation of the functional status of integrin receptors plays a critical role in inflammation and tissue remodeling, as it affects cell adherence and cytokine secretion. We have previously shown that in monocytes the binding of collagen to the alpha 2 beta 1 integrin induces the release of IL-1, an event that is potentiated by binding of fibronectin (Fn) to the alpha 5 beta 1 integrin. In this study, we have investigated the mechanisms leading to this phenomenon. Fn binding to alpha 5 beta 1 induced intracellular signals which increased the alpha 2 beta 1-dependent adhesiveness of monocytes to collagen without modifications of alpha 2 beta 1 expression. By using Abs against the intracellular region of the alpha 5 subunit of the alpha 5 beta 1 receptor, and specific inhibitors of protein kinase C (PKC), we found that the potentiation effect of Fn on monocyte IL-1 production and their adherence to collagen was dependent on an intact alpha 5 subunit cytoplasmic domain, and required PKC activation. Although the alpha 2 beta 1 could be activated by several intracellular second messengers, including protein kinase A and intracellular calcium, the potentiating effect of Fn was mediated only by PKC. These data provide an example of a novel regulatory mechanism: potentiation of beta 1 integrin-mediated events as a result of ligand binding to another integrin of the same class. They also show that the intracellular region of alpha 5 beta 1 plays a critical role in transducing signals generated by ligand binding to alpha 5 beta 1.
Cytokine profile and ultrastructure of intraepithelial gamma delta T cells in chronically inflamed human gingiva suggest a cytotoxic effector function.
Authors: Lundqvist C , Baranov V , Teglund S , Hammarström S , Hammarström M L
Published: December 2022

We have shown that gamma delta T cells in human gingiva have an intraepithelial location and, that in the chronic inflammatory disease periodontitis, the expression of CD45RO and CD8 or CD4 is induced on gamma delta T cells. To study the role of gamma delta T cells in local antibacterial responses, we determined the cytokine profiles of isolated human gingival cells. Different T cell subpopulations, isolated by positive selection with mAb-coated magnetic beads and macrophages, as well as epithelial cells, were analyzed for expression of mRNA for 15 cytokines by reverse transcriptase-PCR. The ultrastructure of gingival gamma delta T cells was also studied. The gamma delta T cells expressed mRNA for IFN-gamma, TNF-alpha, TGF-beta 1, and IL-6. Expression of IFN-gamma was a consequence of inflammation. CD4+ gamma delta T cells expressed IFN-gamma only, whereas CD8+ gamma delta T cells expressed all four cytokines. CD8+ cells expressing IFN-gamma, TNF-alpha, and IL-6 in combination suggest a cytotoxic effector function. Gingival gamma delta T cells contained cytoplasmic electron-dense membrane-bound granules and multivesicular bodies that are ultrastructural characteristics of cytotoxic cells. Epithelial cells from inflamed gingiva expressed HLA-DR, CD1a, CD1c, and heat shock protein 60 on the cell surface. They also expressed mRNA for IL-1 beta, IL-6, IL-8, TNF-alpha, and TGF-beta 1. Thus, epithelial cells may function as accessory cells in immune activation and, at the same time, be target cells for CD8+ gamma delta T cells reactive with CD1 Ag or heat shock protein. These results suggest that gamma delta T cells constitute a first line of defense in gingiva, preventing entrance of pathogens by cytotoxicity against infected and stressed epithelial cells, and by control of epithelial cell growth through secretion of regulatory cytokines.
Adult onset immunodeficiency caused by inherited adenosine deaminase deficiency.
Authors: Shovlin C L , Simmonds H A , Fairbanks L D , Deacock S J , Hughes J M , Lechler R I , Webster A D , Sun X M , Webb J C , Soutar A K
Published: December 2022

Adenosine deaminase (ADA) deficiency is identified here as a cause of adult onset immunodeficiency. Two sisters who noted recurrent, predominantly chest infections in their twenties were found in their thirties to have CD4+ lymphopenia and lymphocyte ADA activity of approximately 5% of the lower limit of normal. Immune function, measured by proliferation of PBMCs in vitro to mitogens and specific Ags, was impaired. Inheritance of a polymorphic marker showed that both patients were heterozygous at the ADA locus. In the paternal allele there was a deletion resulting from homologous recombination between two alu elements that normally flank the first exon and the polymorphic marker. The recombination site was distinct from that in similar deletions described in two infants having severe combined immunodeficiency. This allele is predicted to result in a null phenotype. In the mutant allele inherited from the mother, a C to T transition in a CpG dinucleotide changed the codon for arginine 211, which lies in a conserved sequence close to the active site, to that for cysteine. This mutation has been observed previously in a child in whom the other allele was also a null mutation, but who was diagnosed as having partial ADA deficiency because immune function was apparently normal. The late onset of immunodeficiency in our patients suggests that immune function in children with partial ADA deficiency may deteriorate with time and that ADA deficiency should be regarded as a possible cause of adult onset immune dysfunction of unknown etiology.
IFN-gamma prevents Th2 cell-mediated pathology after neonatal injection of semiallogenic spleen cells in mice.
Authors: Donckier V , Abramowicz D , Bruyns C , Florquin S , Vanderhaeghen M L , Amraoui Z , Dubois C , Vandenabeele P , Goldman M
Published: December 2022

BALB/c mice injected at birth with 10(8) (A/J X BALB/c)F1 hybrid spleen cells develop an autoimmune host-vs-graft (HVG) disease as a result of activation of donor B cells by host CD4+ cells. The antidonor CD4+ cells seem to be Th2-like cells, inasmuch as they are profoundly deficient in IL-2 and IFN-gamma production, but secrete high levels of IL-4 and IL-10. As IFN-gamma is known to inhibit the development of TH2 cells, we attempted to modulate HVG disease by injecting rIFN-gamma. First, we found that 10 micrograms of rIFN-gamma given on days 1 and 3 after birth reduced the serum hyper-IgE of HVG mice by 90% and the serum hyper-IgG1, by 70%. In addition, rIFN-gamma administration significantly decreased the anti-DNA IgG1 titers and prevented the occurrence of anti-glomerular basement membrane and anti-laminin IgG1 Abs as well as the formation of immune deposits in renal glomeruli. These effects were not caused by the abrogation of chimerism, as indicated by the persistence of donor-type B cells in lymph nodes and of Igs bearing donor allotype in serum. MLC experiments indicated that the major effect of early rIFN-gamma administration was to restore the production of IL-2 and IFN-gamma by donor-specific T cells while these cells still secreted significant amounts of IL-4 and IL-10. Unresponsiveness of antidonor cytolytic T cells was not influenced by rIFN-gamma. We conclude that rIFN-gamma prevents the TH2-type response induced by the neonatal injection of semiallogeneic spleen cells and the associated pathology.
Activation and serine phosphorylation of the p56lck protein tyrosine kinase in response to antigen receptor cross-linking in B lymphocytes.
Authors: Gold M R , Chiu R , Ingham R J , Saxton T M , van Oostveen I , Watts J D , Affolter M , Aebersold R
Published: December 2022

We show that cross-linking the B cell AgR with anti-Ig Abs activates p56lck (Lck) in both the immature B cell line WEHI-231 and mature resting B cells from mouse spleen. Anti-Ig-stimulated Lck activity peaked after 1 to 2 min, but remained elevated for at least 15 min. Consistent with the proposed role for src family tyrosine kinases in AgR signaling, we found that Lck could phosphorylate the cytoplasmic tails of the Ig-alpha and Ig-beta components of the B cell AgR in vitro. Lck phosphorylated both of the tyrosines in the Ig-beta AgR homology motif and one of the two tyrosines in the Ig-alpha AgR homology motif. Finally, we show that AgR ligation in B cells caused a significant portion of the Lck to migrate with an apparent molecular mass of 60 kDa on SDS-PAGE gels. Conversion of p56lck to p60lck was maximal at 5 to 15 min, at which times Lck activity in the cells was decreasing. This Lck "band shift" has been observed previously in activated T cells and correlates with phosphorylation of Lck at serine 59. We show that the 60-kDa form of Lck induced by AgR cross-linking in B cells is also phosphorylated at serine 59. Phosphorylation of Lck at this site in vitro decreases its activity. Thus, in B cells, AgR cross-linking activates Lck and subsequently activates a kinase that phosphorylates Lck at serine 59, a potential negative regulatory site.
Developmentally ordered appearance of a functional Fc gamma receptor on TCR V gamma 3 thymocytes. Evidence for stimulation-induced expression.
Authors: Leclercq G , Plum J
Published: December 2022

In the present report, we show that the Fc gamma receptor (Fc gamma R) becomes expressed on mature TCR V gamma 3 thymocytes, but it is absent on immature TCR V gamma 3 cells. Both Fc gamma RIII are Fc gamma RIII are expressed. The in vivo expression of the Fc gamma R on mature TCR V gamma 3 thymocytes coincides with an activated phenotype of the cells. In vitro, anti-CD3 stimulation of Fc gamma R-negative, immature TCR V gamma 3 thymocytes results in the expression of the Fc gamma R. The Fc gamma R of short-term IL-2-cultured, mature TCR V gamma 3 thymocytes is functional in an Ab-mediated cytotoxicity assay. In addition, it is shown that cross-linking of the Fc gamma R induces an increase in cytoplasmic Ca2+. Collectively, our findings show that mature TCR V gamma 3 thymocytes express a functional Fc gamma R; evidence is presented that this could be a result of in vivo activation of these cells.
Mechanisms of dichotomous action of IL-2-Pseudomonas exotoxin 40 (IL-2-PE40) on cell-mediated and humoral immune response.
Authors: Volk H D , Müller S , Yarkoni S , Diamantstein T , Lorberboum-Galski H
Published: December 2022

IL-2-PE40 is a chimeric protein composed of human IL-2 genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain. The immunosuppressive efficacy of IL-2-PE40 was demonstrated in several experimental murine transplant and autoimmune models. However, some observations suggested that IL-2-PE40 could not inhibit the humoral response. In this report, we describe the dichotomous effects of IL-2-PE40 on humoral and cell-mediated immune response in a simple, well characterized in vivo model. Although IL-2-PE40 inhibited the cell-mediated delayed type hypersensitivity reaction to SRBC, it increased the humoral immune response to the same Ag. To understand the mechanism of dichotomous action of IL-2-PE40 on the immune response, IL-2R-bearing T cells were treated with IL-2-PE40 in vitro and the cytokine expression was studied at mRNA and protein level. Similar to IL-2, IL-2-PE40 promoted the expression of T helper 1-like (IFN-gamma) as well as T helper 2-like (IL-4, IL-10) cytokines. These in vitro studies show that IL-2-PE40 can induce signal transduction in activated T cells through the IL-2R before exerting its cytotoxic effect. In contrast to DTH reaction, humoral immune response requires T cell help only for a limited period. Therefore, the short-term stimulation of T helper cells by IL-2-PE40 may be sufficient in vivo to mediate a B cell response in the local environment, whereas the DTH reaction and other cell-mediated immune responses are inhibited by the toxin moiety of the chimeric protein.
Genomic structure, characterization, and identification of the promoter of the human IL-8 receptor A gene.
Authors: Sprenger H , Lloyd A R , Meyer R G , Johnston J A , Kelvin D J
Published: December 2022

Two unique but homologous receptors for the neutrophil chemoattractant, IL-8 have been cloned (designated IL-8RA and IL-8RB), each of which binds IL-8 with high affinity. IL-8RA mRNA expression was found to be regulated by granulocyte-CSF and LPS. In an attempt to understand the tissue-specific expression and to identify transcriptional regulatory elements, we have cloned, sequenced, and characterized the human IL-8RA gene. A lambda-DASH clone encoding the entire human IL-8RA gene was isolated by screening a genomic library with a PCR-generated cDNA. After mapping, subcloning, and sequencing several restriction fragments, a 9.2-kb continuous DNA sequence was obtained. As the sizes of the published cDNA (1.9 kb) and the mRNA determined by Northern blot analysis (2.1 kb) were not in agreement, a full-length cDNA was cloned by using a modified rapid amplification of cDNA ends technique. We identified a 5'-untranslated region of 119 bp. After comparison with the genomic sequence, we found the gene consisted of two exons interrupted by an intron of 1.7 kb. A 1050-bp ORF was encoded entirely in the second exon together with a 834-bp 3'-untranslated region. The immediate GC-rich 5'-flanking region upstream of exon 1 could serve as a constitutively active promoter in chloramphenicol-acetyl-transferase-expression assays. Expression analysis of additional upstream regions suggested the presence of silencer elements between positions -841 and -280. In conclusion, cloning a full-length cDNA permitted us to clone the human IL-8RA gene, identify the genomic structure, and characterize the promoter region.
Lack of expression of the tryptase mouse mast cell protease 7 in mast cells of the C57BL/6J mouse.
Authors: Ghildyal N , Friend D S , Freelund R , Austen K F , McNeil H P , Schiller V , Stevens R L
Published: December 2022

Although the steady-state level of the mouse mast cell protease (mMCP) 7 transcript is below detection in the serosal and mucosal mast cells of the BALB/cJ mouse, the IL-3-dependent, bone marrow-derived mast cells (mBMMC) of this strain and four other strains contain a high steady-state level of the mMCP-7 transcript. To further analyze the expression of this mast cell tryptase, a mMCP-7-specific IgG was obtained by immunizing a rabbit with a 19-residue synthetic peptide that corresponds to its unique amino acid sequence at residues 160 to 178 (anti-mMCP-7(160-178). In a SDS-PAGE/immunoblot analysis of lysates of BALB/cJ mBMMC, anti-mMCP-7(160-178) IgG recognized a diffuse 31- to 36-kDa protein, which shifted to a sharp 27-kDa protein after treatment with N-glycanase. As assessed immunohistochemically, mMCP-7 protein is present not only in the secretory granules of BALB/cJ mBMMC, but also in the ear mast cells of this strain. In contrast, the ear mast cells of the C57BL/6J mouse do not contain detectable levels of mMCP-7 protein, although the ear mast cells of both mouse strains contain mMCP-5 protein. Because mMCP-7 mRNA and protein also were not detected in mBMMC from the C57BL/6J mouse, the failure of the ear mast cells of this strain to express mMCP-7 is most likely a consequence of an intrinsic abnormality in the mast cell-committed progenitor cells themselves, or in the bone marrow microenvironment that induces its mast cell progenitor cells to express this tryptase.
Endotoxin-induced early gene expression in C3H/HeJ (Lpsd) macrophages.
Authors: Manthey C L , Perera P Y , Henricson B E , Hamilton T A , Qureshi N , Vogel S N
Published: December 2022

C3H/HeJ (Lpsd) macrophages have been shown to respond to certain LPSs, especially from rough mutant bacteria. C3H/OuJ (Lpsn) macrophages are induced by wild-type LPS, rough LPS, or lipid A to express many genes, including TNF-alpha, TNFR-2, IL-1 beta, IP-10, D3, and D8. C3H/HeJ macrophages failed to induce any of these genes when cultured with wild-type LPS or synthetic lipid A, even when pretreated with IFN-gamma. However, rough mutant Salmonella minnesota Ra, Rc, and Rd LPS, and Escherichia coli D31 m3 Rd LPS induced Lpsd macrophages to express a subset of genes within the gene panel. Because bioactive preparations contained trace quantities of endotoxin protein(s), a deoxycholate-modified, phenol-water method was used to repurify rough LPS into an aqueous phase, and extract endotoxin proteins into a phenol phase. Repurified LPS failed to stimulate Lpsd macrophages; however, phenol fractions were approximately 10% as potent in Lpsd macrophages as crude rough LPS. Full potency was restored in C3H/HeJ macrophages when aqueous phase LPS and phenol-phase proteins were co-precipitated, suggesting that LPS and endotoxin proteins interact synergistically. Endotoxin proteins alone induced TNF-alpha, TNFR-2, and IL-1 beta, but not IP-10, D3, and D8 genes in both Lpsd and Lpsn macrophages. Tyrosine phosphorylation of three 41- to 47-kDa proteins was induced by endotoxin proteins, but not by LPS, in Lpsd macrophages. Thus, endotoxin proteins seem to activate a signaling pathway(s) that converges (distal to the Lps gene product) with a subset of LPS-signaling pathways.
Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.
Authors: Miskovsky E P , Liu A Y , Pavlat W , Viveen R , Stanhope P E , Finzi D , Fox W M , Hruban R H , Podack E R , Siliciano R F
Published: December 2022

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.
Fetal skin-derived MHC class I+, MHC class II- dendritic cells stimulate MHC class I-restricted responses of unprimed CD8+ T cells.
Authors: Elbe A , Schleischitz S , Strunk D , Stingl G
Published: December 2022

Dendritic cells are very potent, if not the most effective, stimulator cells for the induction of primary T cell immune responses. We have established, from murine fetal skin, growth factor-dependent cell lines with a pronounced dendritic shape and a phenotype similar to that of fetal Langerhans cells (i.e., MHC class I+/II). Functionally, these lines induce a vigorous proliferation of allogeneic, but not syngeneic, CD8+ lymphocytes. T cell blasts thus generated are capable of lysing various target cells in an MHC class I-restricted fashion. Our contention that this skin cell-induced MHC class I-restricted activation of CD8+ lymphocytes occurs in the absence of CD4+ T cells is based on 1) the lack of FACS-detectable CD4+ T cells in the purified CD8+ T cell population, 2) the lack of reactivity of purified CD4+ T cells to MHC class I-disparate fetal skin cell lines, and 3) the inhibition of the fetal skin cell-induced MLR by anti-CD8/MHC class I, but not anti-CD4/MHC class II, mAb. Skin cell-induced activation of unprimed CD8+ T cells was found to be critically dependent on physical contact between stimulator and responder cells and the expression of the costimulatory molecule B7 on fetal skin cell lines. Lines described in this study may represent a powerful tool for studying the molecular events occurring in the induction of MHC class I-restricted primary immune responses, understanding their pathophysiologic role, and perhaps may prove useful for vaccination purposes against selected pathogens.
Recognition of trophoblasts by gamma delta T cells.
Authors: Heyborne K , Fu Y X , Nelson A , Farr A , O'Brien R , Born W
Published: December 2022

The juxtaposition of maternal and fetal tissues in the hemochorial placenta has led to speculation that maternal recognition of fetal Ags present on trophoblasts might play an important role in reproductive biology. We report here for the first time such recognition of trophoblasts by T lymphocyte hybridomas representative of certain cells present in the maternal decidua. Trophoblast recognition is TCR dependent and is mediated by members of the V gamma 1+ subset of gamma delta T lymphocytes, a population that we previously have shown to be associated with heat shock protein-60 reactivity. Recognition occurred in experiments in which trophoblast clones or freshly prepared trophoblasts were used and requires cell-cell interaction. Although the maternal-fetal immune relationship typically has been cast in terms of an allograft, the T cell recognition of trophoblasts as described herein is not MHC-restricted, inasmuch as freshly prepared trophoblasts from beta 2-microglobulin-deficient mice were found to be stimulatory. Furthermore, the trophoblast ligand that mediates this recognition is probably a conserved mammalian molecule, because a human trophoblast cell line is also stimulatory. Our findings suggest a novel form of T cell recognition, and may provide an enhanced understanding of the maternal-fetal immune relationship.
Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes.
Authors: Kim H S , Zhang X , Choi Y S
Published: December 2022

Follicular dendritic cell (FDC)-like cell lines (HK cells) from human tonsils were established to investigate the functional role of FDC in the germinal centers of lymphoid follicles. Although HK cells exhibited CD21, CD23, DRC-1, CD40, VCAM-1, ICAM-1, and HJ2 that were expressed on human tonsilar FDC at early stages of establishment, they lost DRC-1, CD21, and CD23 within 3 days of culture. Morphologic and functional characterization studies suggest that HK cells are quite distinct from fibroblasts. The growth requirement of HK cells is different from the previously reported FDC-like cell lines. Functionally, these cells bound B cells but not T cells, and supported B cell proliferation. The culture supernatants of HK cells had costimulatory activity on the proliferation of anti-mu- or anti-CD40-activated B cells, and the activity dramatically increased after 12-O-tetradecanoylphorbol 13-acetate stimulation. Interestingly, anti-CD3 Ab activated T cell bound to HK cells, inducing the phenotypic changes and the growth of HK cells. The T-HK cell interactions not only involve the well known adhesion ligand/receptor pathways (VLA-4/VCAM-1 and LFA-1/ICAM-1), but also involve CD40-CD40 ligand as shown by inhibitory effect of soluble CD40 (CD40.Fc). The cellular interactions between T and HK cells and cytokine production suggest that activated T cells not only stimulate resting B cells directly, but also support B cell maturation indirectly by stimulating the development of FDC. Hence HK cells may be useful in identifying the surface molecules and cytokines of FDC involved in the GC formation.
Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes.
Authors: Mattern T , Thanhäuser A , Reiling N , Toellner K M , Duchrow M , Kusumoto S , Rietschel E T , Ernst M , Brade H , Flad H D
Published: December 2022

In this paper we describe a new activity of LPS and partial structures: the induction of DNA synthesis and lymphokine production of human T lymphocytes. The LPS-induced T cell proliferation is dose dependent and requires 100 to 10,000 ng/ml of LPS or synthetic lipid A (compound 506) for optimal stimulation. In contrast, the synthetic lipid A precursor Ia (compound 406) is not active but rather antagonizes LPS-induced proliferation. The proliferation is accompanied by the expression of mRNA for the Th1 cell-derived lymphokines IFN-gamma and IL-2, but not for the Th2 lymphokines IL-4, IL-5, or IL-10. Highly enriched T lymphocyte preparations with less than 0.1% monocytes are not stimulated by LPS, showing that monocytes are required for T cell proliferation. Reconstitution experiments show that only monocytes, but not B lymphocytes, are able to support induction of DNA synthesis. Separating LPS-stimulated monocytes from T lymphocytes by a membrane, permeable for cytokines but not for cells, abolishes T cell proliferation. Fixation of monocytes with paraformaldehyde also abrogates their accessory function for T cells. If the monocytes are preincubated for 2 h at 37 degrees C with LPS and then washed, they still are able to induce T cell proliferation in the absence of additional LPS. Our results indicate that human T cells respond in a monocyte-supported manner to LPS exposure by proliferation and lymphokine production. We hypothesize that this reactivity of T lymphocytes to LPS may be of clinical relevance.
MHC control of the naive TCR alpha-chain repertoire.
Authors: Merkenschlager M , Benoist C , Mathis D
Published: December 2022

The naive T cell repertoire is shaped by interactions between developing thymocytes and thymic stroma. Both positive and negative selection involve the clonotypic TCR and MHC molecules carrying self-peptides. Except for the MHC-dependent effects of superantigens on TCR V beta usage; there has been little evidence that the TCR structure of naive T cell varies with the selecting MHC products. To examine this point from another angle, in particular the TCR alpha-chain, we have analyzed alpha-chain usage in a system in which the vast majority of T cells express a transgene-encoded TCR beta-chain, compatible with efficient T cell development on a wide range of MHC haplotypes. Endogenous TCR alpha-chains are thus selected without interference from the forces known to act on TCR-beta, permitting us to observe MHC influences on alpha-chain selection. We have used V alpha-specific Abs to quantitate alpha-chain usage in MHC congenic, MHC recombinant, and MHC transgenic mice and provide evidence that the naive TCR alpha-chain repertoire is under MHC control. The data demonstrate a direct impact of known MHC class II products but also reflect more complex influences, apparently involving other gene products within the MHC. Sequence analysis of differentially selected TCR suggests that selection acts on the entire alpha-chain, including V alpha, J alpha, and the junctional region.