bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased acute-phase response and renal amyloidosis in aged CD2-fas-transgenic mice.
Authors: Hsu H C , Zhou T , Yang P A , Herrera G A , Mountz J D
Published: December 2022

We previously demonstrated that increased Fas expression in T cells of aged CD2-fas transgenic (Fas-Tg) CD-1 mice results in an increased immune response and T cell apoptosis. Surprisingly, despite prevention of T cell immune senescence, the average life span of Fas-Tg mice is comparable with that of nontransgenic (non-Tg) mice. Histopathologic evaluation of tissue sections showed that nearly 50% of the aged (>18-mo-old) Fas-Tg mice developed renal amyloid A amyloidosis, whereas no amyloid deposition was observed in aged non-Tg mice. The amyloid A deposition was observed primarily in glomeruli by using immunohistochemical stains and electron microscopy. The full-length amino acid coding sequence of serum amyloid A2 cDNA in CD-1 mice was identical to that of amyloid A amyloidosis-susceptible BALB/c mice. Although there was no significant difference in steady-state serum amyloid A level in the serum of aged non-Tg and Fas-Tg mice, challenging mice with staphylococcal enterotoxin B resulted in significantly higher serum levels of serum amyloid A on day 2 and IL-6 on days 1 and 2 and a higher magnitude of weight loss on day 7 in aged Fas-Tg mice compared with young mice. These parameters, at the indicated time points, were equivalent between young and aged non-Tg mice. Taken together, our data suggest that prevention of T cell senescence in Fas-Tg mice may be a factor in induction of an excessive acute-phase response triggered by T cell activation. The Fas-Tg mice are a novel model for understanding the immunologic mechanisms leading to secondary amyloidosis.
The IL-4-induced tyrosine phosphorylation of the insulin receptor substrate is dependent on JAK1 expression in human fibrosarcoma cells.
Authors: Wang H Y , Zamorano J , Yoerkie J L , Paul W E , Keegan A D
Published: December 2022

It has been shown that IL-4 induces the tyrosine phosphorylation of JAK1 and JAK3 in the majority of hemopoietic cell types, and JAK2 and TYK2 in several other types. However, the significance of this tyrosine phosphorylation in regulating IL-4 signaling has not been shown. To determine whether JAKs play a role in activating a signal transduction pathway different from the classical JAK/STAT pathway, we analyzed the ability of huIL-4 to stimulate the tyrosine phosphorylation of one of its major cellular substrates, the insulin receptor substrate (IRS). Using human fibrosarcoma cell lines with mutations in JAK1, JAK2, and TYK2, we found that expression of functional JAK1, but not TYK2 or JAK2, is essential for IL-4-induced tyrosine phosphorylation of IRS. We also provide evidence that the IRS pathway is independent of STAT-6, showing that JAK1 is essential for activating a STAT-independent pathway.
T cell adhesion to P-selectin induces tyrosine phosphorylation of pp125 focal adhesion kinase and other substrates.
Authors: Haller H , Kunzendorf U , Sacherer K , Lindschau C , Walz G , Distler A , Luft F C
Published: December 2022

Lymphocyte binding to endothelial surface adhesion molecules is an important early step in inflammation, which is mediated initially by P-selectin and E-selectin. We tested the hypothesis that lymphocyte binding to the selectin adhesion molecules induces intracellular signaling by tyrosine phosphorylation. We used an adhesion assay, which relied on cell binding to chimeric proteins consisting of the extracellular domains for P-selectin and E-selectin. Tyrosine phosphorylation was determined using anti-phosphotyrosine Abs by confocal microscopy and Western blot. Binding to P-selectin induced a significant increase in anti-phosphotyrosine immunoreactivity. The P-selectin effect was time dependent with an early response after 10 min and a maximum effect at 30 min. Western blot showed a time-dependent phosphorylation of two distinct 68- and 125-kDa proteins. These proteins were pp125 focal adhesion kinase (FAK) and paxillin, as shown by immunoprecipitation and colocalization. Phosphorylation of pp125 FAK was time dependent reaching a maximum after 30 min. Incubation with the tyrosine kinase inhibitor genistein, and, to a lesser extent, with the protein kinase C inhibitor staurosporine, resulted in decreased pp125 FAK phosphorylation. Our results are the first to demonstrate that lymphocyte binding to P-selectin induces tyrosine phosphorylation of distinct proteins. Thus, lymphocyte activation may occur already at the initial contact with surface adhesion molecules.
Fc(gamma) receptor cross-linking induces peripheral blood mononuclear cell monocyte chemoattractant protein-1 expression: role of lymphocyte Fc(gamma)RIII.
Authors: Marsh C B , Wewers M D , Tan L C , Rovin B H
Published: December 2022

Immune complexes activate cells by cross-linking leukocyte surface Fc(gamma)Rs. Diseases associated with immune complex deposition, such as rheumatoid arthritis, glomerulonephritis, or idiopathic pulmonary fibrosis, are characterized by compartmentalized monocyte infiltration. The factors that recruit monocytes to these compartments are not well characterized; however, monocyte chemoattractant protein-1 (MCP-1) has been found in areas of tissue injury. To account for these observations we hypothesized that PBMC Fc(gamma)R cross-linking may induce MCP-1 synthesis, which stimulates further monocyte recruitment. To test this hypothesis, PBMC were incubated on increasing concentrations of immobilized human IgG, a stimulus for Fc(gamma)R cross-linking. Immunoreactive MCP-1 was produced in a dose-dependent manner (p < 0.0001). MCP-1 was specifically induced by Fc(gamma)R cross-linking, since immobilized F(ab')2 fragments of human IgG did not activate MCP-1 production. This effect was reproduced by directly cross-linking PBMC Fc(gamma)RIII, but not by cross-linking Fc(gamma)RI or Fc(gamma)RII. PBMC-derived MCP-1 stimulated monocyte chemotaxis that was inhibited by a neutralizing anti-MCP-1 Ab. MCP-1 levels correlated with increased PBMC mRNA expression. Interestingly, Fc(gamma)R cross-linking with either immobilized IgG or anti-Fc(gamma)RIII induced more MCP-1 release from PBMC than from autologous monocytes (p = 0.02). Lymphocytes, the main cell type found in PBMC preparations, did not independently produce a significant amount of MCP-1, but when incubated on immobilized IgG or anti-Fc(gamma)RIII secreted a soluble factor(s) that induced monocyte MCP-1 production. These data suggest that cross-linking PBMC Fc(gamma)R induces the production of bioactive MCP-1. This occurs in part at the level of gene transcription and involves a cooperative interaction between monocytes and lymphocytes.
Molecular targets of CD45 in B cell antigen receptor signal transduction.
Authors: Pao L I , Bedzyk W D , Persin C , Cambier J C
Published: December 2022

Expression of the phosphotyrosine phosphatase CD45 is essential for B cell Ag receptor (BCR)-mediated p21ras activation and calcium mobilization. To examine the molecular basis of this requirement, we analyzed signaling events following BCR ligation in CD45-deficient (CD45-) and CD45-reconstituted (CD45+) variants of J558Lmicrom3 cells. Ag stimulation resulted in tyrosine phosphorylation of cellular proteins in both cells. However, the spectrum of proteins phosphorylated in the CD45+ cells was qualitatively and/or quantitatively distinct from that in the CD45- cells. Among the protein tyrosine kinases examined, the Src family kinases Fyn and Blk were inducibly tyrosine phosphorylated and activated by receptor ligation only in CD45+ cells. While Ag-induced Btk tyrosine phosphorylation occurred in both cells, its activation was greatly diminished in the CD45- cells. Analysis of specific effector molecules revealed that tyrosine phosphorylation of Shc, but not rasGAP or Vav, correlated with the unique ability of BCR ligation to trigger p21ras activation in CD45+ cells. BCR-mediated Shc phosphorylation and recruitment of Grb2 depended on CD45 expression. Thus, Shc tyrosine phosphorylation may be the primary CD45-dependent mechanism by which Ag receptors are coupled to the p21ras pathway in J558Lmicrom3. In addition, phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 were tyrosine phosphorylated upon Ag stimulation in CD45- cells, despite much reduced inositol trisphosphate production and lack of calcium mobilization. These findings suggest that CD45 may modulate events other than PLCgamma phosphorylation, which regulate phosphoinositide hydrolysis and the calcium mobilization response following BCR ligation.
Optimal T cell activation by melanoma cells depends on a minimal level of antigen transcription.
Authors: Labarriere N , Diez E , Pandolfino M C , Viret C , Guilloux Y , Le Guiner S , Fonteneau J F , Dreno B , Jotereau F
Published: December 2022

We reported previously that a large fraction of melanoma cell lines induced a suboptimal activation of specific CTL clones, characterized by good tumor cell lysis but no detectable IL-2 production. Using synthetic peptides, we demonstrated recently that this was due to expression of subthreshold levels of appropriate MHC-peptide complexes. We measure here by semiquantitative reverse transcription-PCR the expression of two melanoma Ag (NA17-A and Melan-A/MART-1) mRNAs in 13 melanoma cell lines and analyze the responses to these cell lines of specific HLA-A2-restricted CTL clones. In line with the idea that the density of MHC-antigenic peptide complexes on melanoma cells is a direct function of the Ag's mRNA level, we found that CTL lysis was grossly proportional to this level. We also established that a minimal level of transcription is required for melanoma cells to induce IL-2 secretion. Interestingly, all cell lines that expressed the Ag above this minimal level, either spontaneously or after gene transfection, stimulated the secretion by tumor-infiltrating lymphocyte of IL-2 amounts proportional to Ag expression unless they exhibited a defective expression of intracellular adhesion molecule-1 or LFA-3 molecules or a low expression of the restricting HLA element. These results indicate that optimal activation and therefore, doubtless, full functionality of melanoma-specific CTL clones critically depend on the mRNA level of the Ag in tumor cells and also on a minimal expression of the HLA restriction element, intracellular adhesion molecule-1, and LFA-3. These data provide a rationale for a better selection of patients to be included in Ag-specific immunization protocols.
Single immunizing dose of recombinant adenovirus efficiently induces CD8+ T cell-mediated protective immunity against malaria.
Authors: Rodrigues E G , Zavala F , Eichinger D , Wilson J M , Tsuji M
Published: December 2022

The immunogenicity of a recombinant replication defective adenovirus expressing a major malaria Ag, the circumsporozoite (CS) protein (AdPyCS), was determined using a rodent malaria model. A single immunizing dose of this construct induced a large number of CS-specific CD8+ and CD4+ T cells in the spleens of these animals, particularly when given by the s.c. or i.m. route. A single dose of AdPyCS also induced high titers of Abs to Plasmodium yoelii sporozoites in mice. No other form of presentation of the CS protein given as a single immunizing dose, i.e., irradiated sporozoites, recombinant vaccinia, or influenza virus, etc., elicits comparably high numbers of CS-specific CD8+ T cells. The high concentration of CS-specific CD8+ T cells in the spleen was relatively short-lived, decreasing to half of its original value by 4 wk and to one-third at 8 wk after AdPyCS inoculation. The decrease in splenic CS-specific CD4+ T cells was even more rapid. Most importantly, a single dose of inoculation of AdPyCS into mice rendered them highly resistant to sporozoite challenge, resulting in a 93% inhibition of liver stage development of the parasites. This protective effect was primarily mediated by CD8+ T cells, as shown by depletion of this T cell population, while depletion of the CD4+ T cell population had only a minor effect on anti-plasmodial activity. Moreover, the inoculation of mice with AdPyCS induces sterile immunity in a significant proportion of mice, preventing the occurrence of parasitemia.
A Ca2+-dependent autoregulation of lipopolysaccharide-induced IL-8 receptor expression in human polymorphonuclear neutrophils.
Authors: Bhattacharya C , Samanta S , Gupta S , Samanta A K
Published: December 2022

IL-8, a potent neutrophil chemotactic agent, is known to be a key mediator in several inflammatory diseases. We found that 10 ng/ml of serum-activated LPS (Escherichia coli) efficiently up-regulated IL-8R on the surface of neutrophils within 30 min of LPS stimulation by 115 to 120% through de novo protein synthesis. After 30 min of LPS stimulation, reduction of IL-8R level was initiated and the normal level was restored within 2 h of LPS interaction. EDTA or EGTA and bestatin separately inhibited the receptor down-regulation by 98%, indicating the involvement of metalloprotease(s), more specifically an aminopeptidase in the process. Induction and subsequent reduction of IL-8 binding in serum-activated LPS-stimulated cells have been demonstrated in autoradiography. Intracellular Ca2+ level in these stimulated neutrophils was increased and decreased with alteration of IL-8R level. Although IL-8 binding was drastically reduced, the total IL-8R level, as detected by anti-IL-8R Ab measured by 125I-labeled anti-rabbit IgG, remained almost unaltered, indicating that minimal proteolysis occurred in IL-8R. Anti-IL-8R Ab and IL-8 itself could prevent this down-regulation significantly, suggesting that the susceptible epitope(s) might be in the IL-8 binding domain of the receptor. Under Ca2+-depleted conditions, the proteolysis was inhibited, which was accelerated upon addition of 1 mM of CaCl2. The study demonstrates that LPS-induced up-regulation of IL-8R leads to amplified IL-8-mediated biologic responses of neutrophils that are restored to normal level by activation of a Ca2+-dependent aminopeptidase. This may be useful for understanding the regulation of LPS-mediated inflammatory responses of neutrophils during bacterial infection.
Expression and release of the monocyte lipopolysaccharide receptor antigen CD14 are suppressed by glucocorticoids in vivo and in vitro.
Authors: Nockher W A , Scherberich J E
Published: December 2022

The effect of glucocorticoid (GC) treatment on expression and release of the monocyte cell surface LPS receptor Ag CD14 was studied in vivo and in vitro. In patients with acute inflammatory diseases receiving GC pulse therapy serum concentrations of soluble CD14 and CD14 expression by peripheral blood monocytes decreased significantly. The LPS-binding capacity correlated positively with the amount of cell surface CD14 by human blood monocytes. In vitro, a time- and dose-dependent effect of GC preparations on monocyte membrane and soluble CD14 by cultured peripheral blood monocytes was found. Incubation with 2 x 10(-8) M prednisolone down-regulated cell surface CD14 after 72 h, and 2 x 10(-7) M suppressed CD14 expression even after 24 h. Prednisolone also decreased release of the soluble CD14 Ag, where a 10-fold higher GC concentration was required for a significant suppression compared with membrane CD14 during culture. Expression of other monocyte membrane Ags were either unchanged (CD33, CD35), diminished (CD13, CD89), or increased (CD32) by GC, indicating no general down-modulation of cell surface Ag expression. Preincubation with glucocorticoids for 24 h significantly down-regulated CD14 expression during subsequent steroid-free culture for at least 7 days. In cultured monocytes, the LPS-induced increase of membrane and soluble CD14 was markedly but not completely inhibited by prednisolone. Therefore, GC treatment suppresses the up-regulation of the LPS receptor during endotoxin challenge, and likewise, the IL-1 secretion after LPS stimulus was significantly diminished. Taken together, the suppression of the monocytic cell surface and soluble endotoxin receptor CD14 by GC may contribute to the increased risk of infections in patients undergoing steroid therapy.
Chemoattractant cross-desensitization of the human neutrophil IL-8 receptor involves receptor internalization and differential receptor subtype regulation.
Authors: Sabroe I , Williams T J , Hébert C A , Collins P D
Published: December 2022

Human neutrophils undergo rapid homologous receptor desensitization following repeated stimulation with chemoattractants such as IL-8, C5a, and FMLP. It has also been demonstrated that cross-desensitization among these chemoattractant receptors occurs. We investigated the mechanisms underlying the cross-desensitization of responses to IL-8 induced by pretreatment with FMLP or C5a. In [125I]-labeled IL-8 binding studies we found that the cross-desensitization induced by FMLP or C5a was associated with a subsequent reduction in IL-8 binding to neutrophils. There was no recovery of [125I]-labeled IL-8 binding on removal of the C5a or FMLP pretreatment. FACS analysis using mAbs specific for the two IL-8R subtypes showed differential regulation of IL-8R A and IL-8R B cell surface expression after chemoattractant pretreatment. Homologous desensitization by IL-8 resulted in internalization of IL-8R A and IL-8R B, but only IL-8R A was completely re-expressed after removal of agonist. FMLP stimulation led to a substantial loss of IL-8R B from the cell surface, whereas C5a stimulation induced only a partial loss. In both cases there was no re-expression of IL-8R B on removal of the chemoattractant stimulation. C5a and FMLP did not affect IL-8R A expression. Calcium mobilization studies using melanoma growth stimulatory activity and IL-8 suggest that a sustained loss of IL-8R B may play a part in maintaining FMLP-induced IL-8R cross-desensitization. Chemoattractant-induced cross-desensitization of neutrophils may be of importance in regulating neutrophil accumulation during the inflammatory response in vivo.
Roles of IL-4 and IL-12 in the development of lupus in NZB/W F1 mice.
Authors: Nakajima A , Hirose S , Yagita H , Okumura K
Published: December 2022

Development of either Th1 or Th2 cell subsets has profound immunologic consequences, either pathogenic or protective, in several autoimmune diseases. However, it remains unclear which subset of Th cells plays a more critical role in lupus. In this study, we examined IL-4 and IL-12, which play decisive roles in the development of Th2 and Th1, respectively, in the IgG autoantibody production and development of lupus nephritis in NZB/W (B/W) F1 mice. Transfer of either IL-4- or IL-12-stimulated splenocytes from 5-mo-old B/W F1 mice into B/W F1 mice of the same age enhanced the production of IgG anti-dsDNA Ab. Consistently, administration of mAb against either IL-4 or IL-12 before the onset of lupus could inhibit the production of IgG anti-dsDNA Ab. However, only anti-IL-4 mAb was effective in preventing the onset of lupus nephritis. This discrepancy appeared to be explained by the differential effect on the production of IgG3-type autoantibody and TNF production. Interestingly, when combined, anti-IL-12 mAb abrogated the beneficial effect of anti-IL-4 mAb. These results indicate that both Th2 and Th1 contribute to the IgG autoantibody production, and IL-4 and IL-12 play key roles in the complexity of cytokine regulation in the pathogenesis of autoimmunity in lupus, but the former is more critical.
The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C.
Authors: Nelson D R , Marousis C G , Davis G L , Rice C M , Wong J , Houghton M , Lau J Y
Published: December 2022

Cellular immune responses, particularly those mediated by CD8+ CTL, may be important in the pathogenesis and control of hepatitis C virus (HCV) infection. To define the role of HCV-specific CTL in chronic hepatitis C, HCV-specific CTL activity in liver and peripheral blood was assessed in 35 patients with chronic HCV infection and 5 non-HCV controls. HCV-specific CTL activity of expanded CD8+ cells was evaluated against autologous lymphoblastoid cells transduced with recombinant vaccinia virus vectors expressing HCV genotype 1a Ags. CTL activity was detected in unprimed bulk-expanded CD8+ cells derived from the liver in 16 of the 35 patients, but not in peripheral circulation. Three patients infected with non-type 1 HCV were found to have HCV-specific CTL activity against HCV type 1a epitopes, all directed toward HCV core region. Compared with patients without detectable HCV-specific CTL activity based on our assay, those exhibiting CTL activity had lower levels of viremia (p < 0.01 for both branched DNA version 1.0 and 2.0 assays) and more active disease, as reflected by a higher histologic activity index (p = 0.006) and serum alanine aminotransferase levels (p = 0.03). It is concluded that 1) with nonspecific stimulation, HCV-specific CTL activity is found more commonly in the liver than in peripheral circulation, suggesting a tissue-specific localization with HCV-specific CTL and/or its precursors; 2) cross-genotype CTL activity exists, especially toward HCV core, which is relatively conserved across genotypes; and 3) patients with intrahepatic HCV-specific CTL activity had lower levels of viremia and more active liver disease.
Regulation of CD28 costimulation in human CD8+ T cells.
Authors: Lloyd T E , Yang L , Tang D N , Bennett T , Schober W , Lewis D E
Published: December 2022

Optimal stimulation and prevention of anergy in T cells requires signaling through the CD28 molecule. During HIV disease progression, CD28 expression is lost, particularly on CD8+ T cells. Because alterations in cytokine production patterns occur during HIV infection, we determined whether CD8+ T cell phenotype or function was affected by cytokine environment. Treatment of CD8+ T cells with IL-4 decreased levels of both CD28 surface expression and message and increased CD8 expression. Furthermore, CD8+ T cells that had down-regulated CD28 had reduced proliferative capacity. The inhibitory effects of CD28 reduction could be compensated either by increased anti-CD3 or by exogenous IL-2, suggesting that the strength of T cell signaling necessary for the production of IL-2 and subsequent proliferation is negatively regulated by IL-4. CD8+ subpopulations with differential CD28 expression produced different patterns of cytokines, particularly IL-2 and IFN-gamma. Furthermore, CD8+ T cells that had reduced CD28 levels but made their own IL-2 were able to proliferate in response to TCR stimulation. These results suggest that loss of CD28 expression and CD8 T cell function can be regulated by the cytokine environment, which may be altered during HIV disease progression. Whether the dysfunction of CD8+ T cells in HIV infection occurs by such a mechanism is the subject of future investigation.
Surface expression of hemopoietic cell phosphatase fails to complement CD45 deficiency and inhibits TCR-mediated signal transduction in a Jurkat T cell clone.
Authors: Musci M A , Beaves S L , Ross S E , Yi T , Koretzky G A
Published: December 2022

Previous studies have shown that the protein tyrosine phosphatase CD45 is required for initiation of signal transduction through several lymphoid receptors. In contrast, there is increasing evidence that another protein tyrosine phosphatase, hemopoietic cell phosphatase (known as HCP, SHP, PTP1C, SHPTP-1, or PTPN6), is a negative regulator of signaling in hemopoietic cells. To determine the effect of HCP on signal transduction through the TCR, HCP was expressed as a chimeric molecule with extracellular and transmembrane domains of the HLA-A2 molecule (A2/HCP) on wild-type Jurkat T cells and the CD45-deficient variant, J45.01. In this report, we show that expression of A2/HCP, unlike A2 chimeras containing the enzymatic regions of CD45, fails to rescue TCR-mediated signal transduction in J45.01. Furthermore, expression of A2/HCP on wild-type Jurkat T cells results in diminished inositol phosphate production following TCR ligation as well as markedly diminished nuclear factor of activated T cells promoter activity. Surprisingly, however, TCR-mediated tyrosine phosphorylation of phospholipase C gamma 1 remains intact in the Jurkat cells expressing the A2/HCP chimera. These experiments provide further evidence that HCP can serve a negative regulatory role in receptor-mediated signaling in immune cells. Additionally, our studies suggest that surface expression of HCP in T cells may provide a means to identify phosphotyrosine-containing proteins that are required for coupling signaling pathways initiated by ligation of the T cell Ag receptor.
Type 2 helper T cell-type cytokines and the development of "infectious" tolerance in rat cardiac allograft recipients.
Authors: Onodera K , Hancock W W , Graser E , Lehmann M , Sayegh M H , Strom T B , Volk H D , Kupiec-Weglinski J W
Published: December 2022

CD4-targeted therapy with a nondepleting RIB-5/2 mAb abrogates accelerated (< 36 h) rejection in presensitized LEW rats and results in permanent acceptance of LBNF1 cardiac allografts in conjunction with the features of infectious tolerance. This study examined the role and functional significance of the Th1 and Th2 cytokine network and systemic host allospecific Ab (allo-Ab) responses in the development of the infectious tolerance pathway in this model. Long term survival of cardiac transplants in rats treated with the tolerizing RIB-5/2 mAb regimen was accompanied by profound depression of Th1 (IL-2 and IFN-gamma) and Th2 (IL-4, IL-10) cytokines at the graft site, as shown by competitive template reverse transcription-PCR and immunohistochemistry. In contrast, the expression of Th2-type cytokines was selectively up-regulated after transfer of infectious tolerance by spleen cells into new generations of primary and secondary test recipients. Donor-specific circulating IgM allo-Ab responses were diminished throughout, and the switch from IgM to IgG allo-Ab was completely prevented in tolerant hosts, as shown by flow cytometry. The demonstration that treatment with cytolytic anti-CD4, but not anti-CD8, mAb recreated rejection of test cardiac allografts with simultaneous down-regulation of IL-4 mRNA/protein expression underlines the importance of this cytokine in the development of infectious tolerance. Hence, this report documents distinct cytokine elaboration patterns in animals tolerized by CD4-targeted therapy compared with those rendered tolerant by putative regulatory Th2-like cells. The mechanism of tolerance in anti-CD4 mAb-treated hosts appears distinct from that operating in the absence of mAb, when the tolerant state is being transferred in an infectious manner to new cohorts of test recipients.
Function of lymphocyte homing-associated adhesion molecules on human natural killer and lymphokine-activated killer cells.
Authors: Uksila J , Salmi M , Butcher E C , Tarkkanen J , Jalkanen S
Published: December 2022

We have analyzed the ability of fresh and rIL-2-activated human NK cells to interact with high endothelial venules (HEV) that are known to support physiologic lymphocyte extravasation, and examined the role of different adhesion molecules in this process. In in vitro HEV-binding assays, NK cells bound to both peripheral lymph node (pLN) and mucosal HEV. Activation by rIL-2 slightly decreased adherence to pLN HEV, but increased adherence to mucosal high endothelium. Markedly fewer NK cells than PBL expressed L-selectin, and the expression was diminished further upon treatment with rIL-2. Inhibition studies showed, however, that L-selectin was the most important single molecule to mediate adhesion to pLN HEV. Binding to mucosal HEV was mediated mainly by CD44 and alpha 4 integrin, and the expression level of these molecules was increased by rIL-2, paralleling the results in HEV-binding assays. Higher m.w. forms of CD44, representing differentially glycosylated/variant forms of CD44, were more abundant on large granular lymphocytes than on unseparated PBL. We conclude that, despite weak recirculatory capacity, NK cells or a subpopulation of NK cells with the correct adhesion molecules can interact with and bind to high endothelial cells. Lymphokines can modulate the expression of adhesion molecules that NK cells utilize for HEV binding. Our results suggest that activation of NK cells with IL-2 may facilitate the extravasation of lymphokine-activated killer cells, especially to mucosal sites, whereas homing to peripheral lymphoid tissues may be diminished. This should be taken into consideration when procedures for lymphokine-activated killer cell immunotherapy are planned.
The 14.1 surrogate light chain promoter has lineage- and stage-restricted activity.
Authors: Donohoe M E , Blomberg B B
Published: December 2022

The 14.1 surrogate light chain protein is expressed on human pre-B lymphocytes in association with Vpre-B and the mu Ig heavy chain to form the pre-B receptor. The 14.1 chain has also been called the lambda (lambda)-like (LL) protein and is homologous to murine lambda5. The 14.1(IGLL1) gene is expressed in a lineage- and stage-restricted manner. To understand the molecular mechanism of the 14.1 gene tissue- and stage-specific expression, we analyzed the 5'-flanking region and characterized the promoter for this gene. In this report, we identify two DNase I-hypersensitive sites located at 2.6 kilobases (HSS 1) and 0.2 kilobases (HSS 1) upstream of 14.1 exon 1. These hypersensitive sites are present in pre-B lymphocyte cell lines, but absent in mature B and T cell lines. We have used RNase protection analysis to localize the 5' major transcriptional start site and rapid amplification of 5' cDNA ends to identify multiple start sites within the TATA-less, GC-rich 14.1 promoter. The region encompassing HSS 2 was analyzed for promoter activity. Transfection of cell lines with a series of truncated segments of the 5' flanking region linked to the luciferase reporter gene revealed that the 14.1 promoter is lineage- and stage-specific, and we localized this activity to positions +150 to +227 relative to the 5' major transcriptional start site.
Regulation of class I-restricted epitope processing by local or distal flanking sequence.
Authors: Yellen-Shaw A J , Eisenlohr L C
Published: December 2022

Influenza A/Puerto Rico/8/34 nucleoprotein (NP) contains an H-2Kd-restricted CD8+ T cell (T CD8+) epitope spanning amino acid residues 147-155. It was previously demonstrated that expression of NP147-155 and NP147-158 in isolation via "minigene"/recombinant vaccinia virus (vac) technology leads to sensitization of target cells for NP-specific killing while expression of 147-158 lacking the arginine at position 156 (termed here as 147-155TG) does not. The presentation block was overcome by placing this fragment into the context of full length NP. We show that addition of a single amino acid, Met159, to the C terminus of the blocked peptide (creating 147-155TGM) restores presentation. Presentation of 147-155TGM was not due to trimming in the exocytic compartment, consistent with severe limitations on C-terminal trimming activity in this location. Rescued presentation was also achieved when the blocked construct was extended in the N-terminal direction only, but in this case more than 55 amino acids of flanking sequence were required. The transition to presentation was abrupt, with 91-155TG and shorter constructs showing little or no detectable presentation and 90-155TG showing full level presentation. Presentation could not be attributed to acquisition of conventional targets for ubiquitination since mutation of all Lys residues, to which the ubiquitin moiety is conjugated, does not abrogate presentation. Rescued presentation was not inhibited by the peptide aldehyde N-acetyl-L-leucinyl-L-leucinal-L-norleucinal, suggesting that the added elements may be recruiting nonproteasomal activity. We have therefore identified and begun to characterize protease targeting of regulatory elements, both local and distal to an epitope, which strongly influence the ability of the epitope to be excised.
C5a-dependent up-regulation in vivo of lung vascular P-selectin.
Authors: Mulligan M S , Schmid E , Till G O , Hugli T E , Friedl H P , Roth R A , Ward P A
Published: December 2022

Acute lung injury in rats following systemic activation of complement by i.v. infusion of cobra venom factor (CVF) is known to be P-selectin dependent. In the current studies, infusion of CVF caused the appearance in plasma of C5a (as revealed by ELISA analysis) together with neutrophil chemotactic activity, which was totally blocked by addition in vitro of anti-rat C5a. Using a detector 125I-labeled antibody to rat P-selectin, we have demonstrated quantitative up-regulation in vivo of lung vascular P-selectin in a time-dependent manner after infusion of CVF. This up-regulation was almost completely blocked by prior complement depletion or by the infusion of anti-rat C5a. Platelet depletion did not affect CVF-induced up-regulation of lung vascular P-selectin, indicating that platelets were not the source of P-selectin. These results demonstrate that complement and, specifically, C5a are necessary for up-regulation of lung vascular P-selectin after systemic activation of complement.
Mechanisms of action of antimalarials in inflammation: induction of apoptosis in human endothelial cells.
Authors: Potvin F , Petitclerc E , Marceau F , Poubelle P E
Published: December 2022

Antimalarials are beneficial therapeutic agents in systemic lupus and rheumatoid arthritis. These autoimmune diseases have abnormally low apoptosis of inflammatory cells. Both disorders have an abnormal angiogenesis. In the present report, antimalarials were demonstrated to selectively increase apoptosis of HUVECs in vitro. A 24-h exposure to 50 or 150 microM of the drugs was associated with a significant loss of substrate-adherent cells. Chloroquine exhibited an inhibitory effect on HUVEC proliferation over 7 days. Programmed cell death in HUVECs rendered nonadherent by chloroquine was confirmed by the induction of DNA fragmentation in floating cells. Northern blot analysis revealed a rapidly increased expression of the bcl-x(s) gene without any change in the expression of the bcl-2 gene, indicating that HUVECs under chloroquine were undergoing apoptosis. The onset of the apoptotic cascade in HUVECs appeared shortly after the addition of chloroquine. The effect of chloroquine on apoptosis was distinct from acute cell lysis and was restricted to HUVECs. Antimalarials also induced IL-1alpha production. In parallel, chloroquine alone did not increase the expression of IL-6. Anti-IL-1alpha Ab or IL-1Ra only marginally reversed chloroquine-induced depression of proliferation for the low drug concentration, but not the massive cell death effect at and above 50 microM. Taken together, these data may indicate that antimalarials repress angiogenesis. The autocrine mechanism involving IL-1alpha accounts only for a minor fraction of the full antiendothelial effect of chloroquine, which is mainly dependent on apoptosis.
Analysis of human common bile duct-associated T cells: evidence for oligoclonality, T cell clonal persistence, and epithelial cell recognition.
Authors: Probert C S , Christ A D , Saubermann L J , Turner J R , Chott A , Carr-Locke D , Balk S P , Blumberg R S
Published: December 2022

The phenotype of T cells associated with the common bile duct (CBD) is unknown. We investigated the hypothesis that they behave like other intraepithelial lymphocytes (IEL). Thus, we sought to determine the phenotype, TCR repertoire, and epithelial recognition of T cells obtained during endoscopic retrograde cholangiopancreatography. Three subjects were studied: two with primary sclerosing cholangitis and one normal control. After establishing a short-term T cell line, cells were 1) stained with mAbs for flow cytometric analysis, 2) analyzed for TCRB chain transcript expression, and 3) used as effector cells for cytotoxicity and proliferation. Flow cytometry revealed that for all the subjects 98% of the T cells were TCR-alpha beta-positive. Immunohistology of the CBD showed that the epithelium and lamina propria contained significant numbers of CD3+ CD43+ CD45RO+ lymphocytes. Complementarity-determining region 3 length displays suggested that the CBD-derived lines were oligoclonal. This was confirmed by cloning and random sequencing of PCR amplification products using TCRBV region family-specific primers; TCRB chain sequences were reiterated in all transcripts analyzed. In one case, two expanded TCRB clones could be identified that were persistent in the bile duct over a 1-yr period. The CBD-derived lines were cytolytic in a redirected lysis assay and caused cytolysis of an intestinal epithelial cell line (Caco-2). This recognition was likely preferential for intestinal epithelial cells, since a CBD-derived line exhibited proliferation to two intestinal epithelial cell lines (HT-29 and Caco-2) but not three other lines (HepG2, human foreskin fibroblast, and KD). We conclude that the CBD contains IELs that share several characteristics with intestinal IELs.
Human T cell responses induced by vaccination with Mycobacterium bovis bacillus Calmette-Guérin.
Authors: Ravn P , Boesen H , Pedersen B K , Andersen P
Published: December 2022

Many aspects of the widely used bacillus Calmette-Guérin (BCG) vaccine against tuberculosis are still the subject of controversy. There is a huge variation in efficacy from one clinical trial to another and no relationship between vaccine-induced skin test conversion and subsequent protection. We have studied in vitro cell-mediated immune responses primed by BCG vaccination in 22 healthy Danish donors with different levels of in vitro purified protein derivative (PPD) reactivity before vaccination. The study demonstrated a markedly different development of reactivity to mycobacterial Ags depending on the prevaccination sensitivity to PPD. Previously sensitized donors mounted a potent and highly accelerated recall response within the first week of BCG vaccination. Nonsensitized donors, in contrast, exhibited a gradually increasing responsiveness to mycobacterial Ags, reaching maximal levels between day 56 and 365 postvaccination. The recognition of different classes of Ags were induced in a stepwise manner: culture filtrate Ags were recognized 1 wk postvaccination followed by cell wall, membrane, and the cytosolic Ag fraction. The T cell response primed by BCG vaccination was characterized as a CD4 response with a Th1-like cytokine pattern and substantial levels of Ag-specific cytotoxicity. The specificity of the T cell response generated was broad and directed to a range of culture filtrate Ag fractions. The study shows that BCG vaccination of previously nonsensitized donors can provide important data on potentially protective immune responses in humans and suggest a careful evaluation of prevaccination sensitivity when investigating vaccine-induced immunity.
C-X-C and C-C chemokines are expressed in the cerebrospinal fluid in bacterial meningitis and mediate chemotactic activity on peripheral blood-derived polymorphonuclear and mononuclear cells in vitro.
Authors: Spanaus K S , Nadal D , Pfister H W , Seebach J , Widmer U , Frei K , Gloor S , Fontana A
Published: December 2022

The appearance of polymorphonuclear and mononuclear leukocytes in the cerebrospinal fluid (csf) is an important hallmark of bacterial meningitis. Chemokines are candidate mediators of cell migration from blood into the subarachnoid space. Therefore, concentrations of C-X-C and C-C chemokines in the csf of patients with pyogenic meningitis were measured by ELISA. Highly significant elevations of chemokine levels in comparison with noninflammatory csf controls were found for IL-8 (median, 21.6 ng/ml; range, < 0.1 to 191.3), growth-related gene product alpha (median, 5.6 ng/ml; range, < 0.1 to 48.2), monocyte chemotactic protein-1 (median, 26.4 ng/ml; range, < 0.2 to 193.8), macrophage inflammatory protein-1 alpha (MIP-1 alpha; median, 1.8 ng/ml; range, < 0.5 to 18.0), MIP-1 beta (median, 10.6 ng/ml; range, < 0.3 to 84.4), but not for RANTES (regulated upon activation, normal T cell expressed and secreted). The csf of bacterial meningitis were chemotactic for neutrophils and mononuclear leukocytes. Correlation analysis demonstrated a strong association between individual chemokine levels and chemotactic activity mediated by csf. A significant reduction of neutrophil chemotaxis was obtained by anti-IL-8 and anti-growth-related gene product alpha Abs, and a reduction of mononuclear cell migration was achieved by a combination of anti-monocyte chemotactic protein-1, anti-MIP-1 alpha, and anti-MIP-1 beta Abs. Since no significant correlation was found between csf leukocyte counts and chemokine concentrations or chemotactic activity mediated by csf, additional factors influence the extent of pleocytosis in vivo.
Sequestration of p56(lck) by gp120, a model for TCR desensitization.
Authors: Goldman F , Crabtree J , Hollenback C , Koretzky G
Published: December 2022

Ligation of the CD4 receptor by HIV envelope glycoprotein gp120 inhibits T cell activation and signaling through the TCR complex. Recent reports suggest CD4 ligation by gp120 + anti-gp120 Abs uncouples protein tyrosine kinases (PTKs) from the TCR signal-transduction cascade. This finding and other observations led us to hypothesize that the effects of gp120 are mediated through p56(lck), a PTK noncovalently associated with CD4. To test this hypothesis, we first examined the kinetics of gp120/anti-gp120-induced TCR signaling defects in the Jurkat T cell line. Pretreating cells with gp120/anti-gp120 for 1 to 4 h before stimulation prevented TCR-directed PTK activation. Coincident with TCR desensitization, pretreatment with gp120/anti-gp120 also decreased the amount of p56(lck) that could be immunoprecipitated from the Nonidet P-40 detergent-soluble fraction of cellular lysates, while simultaneously increasing the recovery of p56(lck) from the Nonidet P-40 detergent-insoluble fraction (or cytoskeleton). To assess the potential role of the actin in this process, experiments were conducted in the presence of cytochalasin D. Cytochalasin D restored TCR signaling in cells previously desensitized with gp120/anti-gp120 and prevented translocation of p56lck from the Nonidet P-40 detergent-soluble fraction of cell lysates. Furthermore, p56(lck) was found to coimmunoprecipitate with anti-actin. These data suggest that gp120/anti-gp120 may inhibit TCR signaling by sequestering p56(lck) to the cytoskeleton.
Conjugation of human Fc gamma in closed-hinge or open-hinge configuration to Fab'gamma and analogous ligands.
Authors: Stevenson G T , Anderson V A , Kan K S , Worth A T
Published: December 2022

We describe a method for linking human normal Fc gamma1, via stable thioether bonds emerging from its hinge, to any molecule expressing a free sulfhydryl (SH) group. The Fc hinge may be closed by a disulfide (SS) bond or left open. Preparation begins with reduction of the Fc hinge to release four SH groups from its two parallel inter-gamma SS bonds. When the Fc is required in normal closed-hinge configuration, one SH group is alkylated with N-ethylmaleimide under limiting conditions, and one of the inter-gamma SS bonds is reconstituted by SS interchange. The residual SH group, to be used for linking, is left as a 4-dithiopyridyl group suitable for storage. When the Fc is required for conjugation the 4-dithiopyridyl is replaced by a metastable maleimidyl group, which reacts rapidly with SH on the partner molecule to form a tandem thioether link. If the partner is Ab Fab'gamma, linking to cysteines in the Fab'gamma hinge yields derivatives such as FabFc and FabFc2. Chimeric FabFc Abs (mouse Fab'gamma/human Fc gamma1) invoked cellular cytotoxicity in vitro, using human cell lines as targets and human lymphocytes as effectors, whether the Fc hinge was open or closed. The same Abs could kill the same targets by activating human complement, but only when the Fc hinge was closed. Both effector functions were enhanced by the presence of a second Fc in FabFc2. This method of Fc addition can be used to predict the performance of recombinant chimeric Abs and to provide novel molecular geometries.
Predominant appearance of NK1.1+ T cells producing IL-4 may be involved in the increased susceptibility of mice with the beige mutation during Salmonella infection.
Authors: Enomoto A , Nishimura H , Yoshikai Y
Published: December 2022

C57BL/6 mice with the beige mutation (beige mice) showed a high susceptibility to infection with Salmonella choleraesuis compared with C57BL/6 (B6) control mice, as assessed by bacterial number in the peritoneal cavity and the liver. The appearance of NK1.1+ CD3- NK cells was significantly suppressed, while NK1.1+ T cells were increased in the peritoneal cavity of beige mice after Salmonella infection. The expression level of IL-4 mRNA was much higher in freshly isolated NK1.1+ T cells of the infected beige mice, but the expression level of IFN-gamma mRNA was lower than that in the infected control mice. The NK1.1+ T cells produced more IL-4 in response to TCR alphabeta cross-linking, whereas IFN-gamma production upon TCR triggering was significantly impaired in the beige mice compared to that in the control mice. Furthermore, the generation of Salmonella-specific Th1 cells producing IFN-gamma was significantly inhibited in the peritoneal cavity of beige mice after Salmonella infection. However, administration of anti-IL-4 neutralizing mAb to beige mice during salmonellosis restored the generation of Salmonella-specific Th1 cells and decreased the susceptibility to Salmonella. These results suggested that the predominant activation of NK1.1+ T cells producing IL-4 over those producing IFN-gamma may be at least partly involved in the poor generation of Salmonella-specific protective Th1 cells, resulting in the increased susceptibility of beige mice to Salmonella infection.
Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes.
Authors: Tanaka M , Suda T , Yatomi T , Nakamura N , Nagata S
Published: December 2022

Fas ligand (FasL) is a type II membrane protein. Binding of FasL to its receptor, Fas, induces apoptosis. Matrix metalloproteinase cleaves the membrane-bound human FasL to yield the active soluble form. Here, we have produced a large amount of human soluble rFasL using the yeast, Pichia pastoris. The purified rFasL was found to be glycosylated and to exist as a trimer. The rFasL was effective in inducing apoptosis in a Fas-expressing T cell or a fibroblast cell line. The ID50 of rFasL for mouse Fas-expressing T cells was about 0.5 ng/ml. The killing process with rFasL was quick. That is, >80% Fas-expressing mouse cells were killed within 1 h by a saturation concentration of human rFasL. Intravenous administration of 500 microg of human rFasL had a lethal effect in mice. When the mice were pretreated with Propionibacterium acnes, the subsequent injection of 30 microg of human rFasL induced hepatic failure and killed the mice within 24 h. These results indicated that the soluble human FasL is active in inducing apoptosis in vitro and in vivo, and its deleterious effect may be strengthened in patients who are suffering from bacterial infection.