bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of nuclear factor-kappa B and gene expression in human endothelial cells by the common haptens nickel and cobalt.
Authors: Goebeler M , Roth J , Bröcker E B , Sorg C , Schulze-Osthoff K
Published: December 2022

Nickel chloride (NiCl2) and cobalt chloride (CoCl2), two haptens frequently leading to contact hypersensitivity in industrialized countries, induce gene transcription of adhesion molecules ICAM-1, VCAM-1, and E-selectin in endothelial cells. In search of transcriptional mechanisms underlying their gene-inductive effects, we studied the capacity of both haptens to activate nuclear factor (NF)-kappa B, a transcription factor involved in inducible expression of adhesion molecules. Using electrophoretic mobility shift assays, a strong increase of NF-kappa B DNA binding was detected after stimulation of HUVEC with NiCl2 or CoCl2. Supershift analysis using antisera against p50 and p65 confirmed the authenticity of the induced NF-kappa B complex. Neutralizing Abs against TNF-alpha and IL-1 did not inhibit metal hapten-induced activation of NF-kappa B, thus ruling out action via an indirect autocrine pathway. In addition, NiCl2-induced activation of NF-kappa B and adhesion molecule expression was inhibited by the antioxidant pyrrolidine dithiocarbamate, indicating the involvement of redox-dependent mechanisms. Furthermore, NiCl2 was found to induce dose-dependency mRNA production and protein secretion of the NF-kappa B-controlled proinflammatory cytokine IL-6. Our data suggest that distinct allergens represent a new class of so far unknown agents that induce NH-kappa B binding activity that subsequently modulates transcription of cytokine and adhesion molecule genes. Thus, pathomechanisms leading to contact hypersensitivity to NiCl2 and CoCl2 appear to involve not only Ag-specific Langerhans- and T cell-dependent events but also include direct effects on other immunocompetent cells such as the endothelium.
IL-12 stimulates an antiviral type 1 cytokine response but lacks adjuvant activity in IFN-gamma-receptor-deficient mice.
Authors: Schijns V E , Haagmans B L , Horzinek M C
Published: December 2022

Cytokines can be used as adjuvants to enhance and direct protective immune responses induced by vaccines. IL-12, a cytokine that favors the maturation of Th1-type cells and stimulates associated cell-mediated responses was evaluated as immunologic adjuvant for a viral vaccine in a mouse challenge model. When it was administered together with inactivated pseudorabies virus, a herpes simplex virus related alpha-herpesvirus, increased production of IFN-gamma by ex vivo-stimulated splenocytes was observed as well as augmented production of antiviral serum lgG2a. This was associated with increased protection against a lethal challenge infection. Infection of IfN-gamma-neutralizing Ab reduced the increased antiviral resistance in IL-12-treated mice. Also, in mice bearing an inactivated IFN-gamma-receptor gene IL-12 failed to stimulate protection against challenge and the synthesis of antiviral lgG2a. However, in these IFN-gamma-receptor knockout mice, increased antiviral lgG2b levels and enhanced IFN-gamma-secretion, with minimal IL-4 production, by ex vivo-stimulated splenocytes was observed. In wild-type mice administration of recombinant IFN-gamma but not IL-2 mimicked the immune-stimulating activity of IL-12; it is therefore likely that the IL-12 adjuvant activity is largely mediated by physiologic IFN-gamma.
Progression of a vesicular stomatitis virus infection in primary lymphocytes is restricted at multiple levels during B cell activation.
Authors: Schmidt M R , Gravel K A , Woodland R T
Published: December 2022

Small resting B cells do not support a productive vesicular stomatitis virus (VSV) infection, but are induced by B cell activators to become fully permissive for VSV replication. Nonpermissive B cell populations restrict VSV expression at multiple points: transcript levels, translation, and maturation. Unstimulated resting G0 B cells can be infected by VSV and support the synthesis of all VSV mRNAs. Steady-state levels of viral transcripts are selectively enhanced by T cell-derived cytokines to an extent comparable with that seen for cytokine-regulated cellular mRNAs. However, viral proteins are not detected in immunoprecipitates from unstimulated or cytokine-stimulated B cells despite the fact that viral mRNAs are associated with polysomes and can be translated in vitro. This translational block is released by stimulation of infected B cells with mitogenic anti-lg or LPS, or non-mitogenic PMA. VSV virion maturation is also regulated by activation signals, because neither anti-lg nor PMA-stimulated B cells produce high levels of infectious VSV particles. Because anti-lg stimulation supports viral genome replication, maturational arrest is apparently at virus assembly or release. PMA and ionomycin induces changes beyond those seen with anti-lg, because these B cells produce PFUs at levels comparable with those seen with LPS-activated B cells and VSV-permissive cell lines. Activation-dependent regulation of virus expression provides a new paradigm for assessing activator-induced events in B cell differentiation not revealed by previous assessments of proliferation of Ab synthesis.
Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice.
Authors: Szalai A J , Briles D E , Volanakis J E
Published: December 2022

C-reactive protein (CRP) is an acute phase protein with a well known association with infection and other inflammatory conditions. Studies with use of purified CRP in in vitro assays provided early evidence that CRP has antibacterial activity. Subsequently it was shown that passively administered human CRP can protect mice from lethal infection with Streptococcus pneumoniae. In this study, we extend these observations to an in vivo model of host resistance by using human CRP transgenic mice. CRP transgenic mice experimentally infected with S. pneumoniae lived longer and had significantly lower mortality than their nontransgenic littermates. This increased resistance to infection was associated with q 10- to 400-fold reduction of bacteremia. Furthermore, male transgenics exhibited longer survival time than females, and this difference could be attributed to increased expression of CRP by males, which was mediated by testosterone. This study provides the first unequivocal evidence that CRP plays an important role in vivo in host defense against pneumococcal infections, and shows that sex hormones can affect expression of the human CRP transgene in mice.
A crucial role for beta 2 integrin in the activation of eosinophils stimulated by IgG.
Authors: Kaneko M , Horie S , Kato M , Gleich G J , Kita H
Published: December 2022

An IgG-coated surface, such as found on parasites, is one of the most effective physiologic stimuli for eosinophil activation. Recent evidence suggests that cellular adhesion, especially that through the beta 2 integrin, is an important step in cellular activation and accumulation. Therefore, we investigated the role of adhesion molecules in IgG-stimulated eosinophil functions. Cross-linking of eosinophil cytophilic IgG by anti-IgG immobilized to tissue culture plates induced degranulation, whereas soluble anti-IgG did not. Similarly, eosinophils exposed to human IgG immobilized to plates adhered and degranulated; in addition, adherence and subsequent degranulation were inhibited by mAbs to CD18 and CD11b, but not by mAb to CD29, suggesting an important role of beta 2 integrin for these responses. Eosinophil degranulation induced by IgG covalently coupled to Sepharose 4B beads was also inhibited by mAb to CD18. Furthermore, fibrinogen, a ligand for CD11b/18, showed synergistic enhancement of IgG-induced degranulation when it was co-immobilized with IgG to plates. A morphologic study showed that eosinophils, stimulated by immobilized IgG, protrude numerous pseudopods; this morphologic change was inhibited by mAb to CD18. This cellular adhesion seems to affect the early signaling events in eosinophils because the production of inositol phosphates was abolished by mAb to CD18. Interestingly, although superoxide production by eosinophils triggered by immobilized IgG was inhibited by mAb to CD18, superoxide production and morphologic change of neutrophils were not. These results suggest that cell adhesion through CD11b/18 is a crucial step for the activation, signaling, and effector function of eosinophils stimulated by IgG.
Allogeneic bronchoalveolar lavage cells induce the histology of acute lung allograft rejection, and deposition of IgG2a in recipient murine lungs.
Authors: Wilkes D S , Heidler K M , Bowen L K , Quinlan W M , Doyle N A , Cummings O W , Doerschuk C M
Published: December 2022

The immunologic and histologic changes associated with lung allograft rejection are believed to result from the presentation of donor lung alloantigens to recipient lymphocytes resulting in up-regulated Th1 lymphocyte activity. The ability of allogeneic lung immune cells to induce the pathologic and immunologic changes associated with acute lung allograft rejection are unknown. The current study determined whether allogeneic (C57BL/6, I-a(b)) bronchoalveolar lavage (BAL) cells (> or = 97% macrophages), when instilled into the lungs of recipient BALB/c mice (I-a(d)), induced the histology and immunology associated with acute lung allograft rejection. BALB/c mice received BAL cells from either C57BL/6 mice (allogeneic instillate) or BALB/c mice (autologous instillate) or PBS (control) by nasal insufflation weekly for 4 wk. Allogeneic BAL cells resulted in a lymphocytic bronchitis and vasculitis analogous to grade 1 to 2 lung allograft rejection. The mice given allogeneic instillates had a greater percentage of lymphocytes in the BAL fluid than those given autologous instillates. After instillation of allogeneic BAL cells, the Th1 cytokines, IL-2 and IFN-gamma (IFN-gamma), were produced locally in greater quantities and more frequently than Th2 cytokine IL-10. IL-4, another Th2 cytokine, was not detected. The local production of IgG1 and IgG2a, which are dependent on IL-4 and IFN-gamma, respectively, were increased. However, only IgG2a was deposited in the perivascular and peribronchiolar tissues. These data show that installation of allogeneic BAL cells into the airways of recipient mice induced up-regulated Th1 lymphocyte activity and caused the histologic changes associated with lung allograft rejection.
Regulation by corticosteroids of Th1 and Th2 cytokine production in human CD4+ effector T cells generated from CD45RO- and CD45RO+ subsets.
Authors: Brinkmann V , Kristofic C
Published: December 2022

Corticosteroids (CS) are widely used as immunosuppressive and anti-inflammatory agents, but their mechanism of action is not well understood. In this study we analyzed the effects of CS on the growth and differentiation of human CD4+45RO- "naive" and CD4+45RO+ "memory" T cells. To generate effector T cells secreting large amounts of Th1 and Th2 cytokines, FACS-sorted naive and memory subsets were primed and restimulated in vitro via the TCR in the presence of IL-2. CS added during priming reduced clonal expansion of both T cell populations, but the memory subset was 100-fold less sensitive. At lower concentrations, CS favored the development of effector T cells (from both subsets), which upon restimulation produced large amounts of the anti-inflammatory cytokine IL-10, but low amounts of IL-4, IL-5, or IFN-gamma. Interestingly, CS displayed different effects if it was added only during the restimulation of effector T cells. CS were unable to suppress clonal expansion of restimulated effector T cells. In effector T cells derived from the naive subset, CS induced production of IL-4 and IL-10, but blocked production of IL-5 and IFN-gamma. In effector T cells generated from the memory subset, CS blocked production of IL-4, IL-5, and IL-10, but inhibited production of IFN-gamma by only 50%, even if 100-fold higher concentrations of CS were applied. These results indicate that persistent TCR stimulation, e.g., in chronic infection, may reduce the sensitivity of T cells to the antiproliferative effects of CS. Furthermore, the potential of CS to increase or suppress IL-4 and IL-10 production depending on the stage of T cell activation may explain in part the beneficial effects of CS in the treatment of acute inflammation and chronic allergic/asthmatic diseases.
Control of the IL-2 responsiveness of B lymphocytes by IL-2 and IL-4.
Authors: Moreau J L , Chastagner P , Tanaka T , Miyasaka M , Kondo M , Sugamura K , Thèze J
Published: December 2022

A two-step culture system was used to analyze the parameters involved in the acquisition of IL-2 responsiveness by murine B cells. In the first culture, unstimulated, or resting, B cells prepared from spleen of naive animals were challenged during 48 h with IL-2, IL-4, anti-mu, anti-mu+IL-2, anti-mu+IL4, or anti-mu+IL-2 + IL-4. In a second culture, IL-2 responsiveness was followed by measuring either the cell proliferation or the Ig production. It was found that only B cells stimulated by anti-mu+IL-2 were able to respond. The expression of three chains of the IL-2 receptor (IL-2R alpha, IL-2R beta, and IL-2R gamma) was studied by FACS. IL-2R beta and IL-2R gamma were found to be expressed constitutively on resting B cells. IL-2R alpha was induced by anti-mu and anti-mu+IL-2 treatment. Although B cells treated by anti-mu alone are not able to respond to IL-2, they do express an IL-2 binding capacity comparable with B cells treated by anti-mu+IL-2. This paradoxical result suggests that IL-2 has a direct influence on the acquisition of the IL-2 responsiveness. IL-4 exerts a negative effect on the IL-2 response. At the molecular level, IL-4 was found to reduce selectively the IL-2R beta expression at the B cell surface. This effect was confirmed by Northern blot analysis. Maximum expression of the IL-2R beta mRNA is obtained after anti-mu+IL-2 treatment. In the presence of IL-4, expression of the IL-2R beta mRNA is greatly reduced.
IL-1 beta converting enzyme (ICE) is not required for apoptosis induced by lymphokine deprivation in an IL-2-dependent T cell line.
Authors: Vasilakos J P , Ghayur T , Carroll R T , Giegel D A , Saunders J M , Quintal L , Keane K M , Shivers B D
Published: December 2022

Clonal T cells undergo programmed cell death (PCD) or apoptosis when cultured without the appropriate cytokines. The cysteine protease, IL-1 beta converting enzyme (ICE), is implicated in apoptosis based on its structural similarity to the PCD gene, ced-3, in Caenorhabditis elegans and the induction of PCD in fibroblasts transfected with recombinant ICE. We show that the murine IL-2-dependent CTLL T cell line expresses ICE but not IL-1 beta. Interestingly, ICE mRNA and protein levels increase during apoptosis. Yet inhibition of ICE enzymatic activity (> 90%) with either of two cell-permeable ICE inhibitors does not abrogate or delay apoptosis following IL-2 deprivation, as measured by DNA fragmentation and viability. Our results suggest that ICE is not required for apoptosis in lymphokine-deprived T cells.
Sand fly vector saliva selectively modulates macrophage functions that inhibit killing of Leishmania major and nitric oxide production.
Authors: Hall L R , Titus R G
Published: December 2022

The saliva of Phlebotomus papatasi, a sand fly vector for Leishmania major, contains a factor that exacerbates leishmaniasis and may be required for the establishment of infection with Leishmania in nature. We have examined the effect of sand fly saliva on various macrophage functions in vitro. Our data demonstrate that although saliva does not alter uptake of L. major by macrophages, it inhibits the ability of IFN-gamma to activate macrophages to kill the intracellular parasite. This inhibition of parasite killing is observed when both the promastigote and amastigote forms of the parasite are used for infection. Furthermore, this inhibition of parasite destruction correlates with reduction of nitric oxide (NO) production, suggesting that the ability of sand fly saliva to reduce nitrogen oxidation in response to IFN-gamma may be responsible for the inhibitory effect of saliva on intracellular killing of L. major. Finally, despite the fact that saliva inhibits NO production in IFN-gamma-activated macrophages, it does not prevent IFN-gamma from up-regulating class II MHC expression on macrophages. This suggests that the immunosuppressive effect of sand fly saliva on the macrophage is targeted to certain critical, but not all, functions of the cell.
CD40 ligand is required for resolution of Pneumocystis carinii pneumonia in mice.
Authors: Wiley J A , Harmsen A G
Published: December 2022

The role of the CD40-CD40 ligand (CD40L) interaction in resolution of Pneumocystis carinii (PC) pneumonia (PCP) was assessed in a PC-infected severe combined immunodeficiency (SCID) mouse reconstitution model using an anti-CD40L mAb to block CD40L. SCID mice infected with PC were reconstituted with unfractionated spleen cells from immunocompetent donors and given either anti-CD40L mAb or an irrelevant control mAb. Mice given the control mAb resolved the PC infection, whereas those given the anti-CD40L mAb did not. That anti-CD40L mAb also inhibited PC-specific IgG production is consistent with the possibility that cognate CD4+ T cell-B cell interactions are important in PCP resolution. The experiment was then repeated, except that the PC-infected SCID mice were reconstituted with purified CD4+ T cells only. Again, the control mAb-treated group resolved the PCP, whereas mice treated with anti-CD40L mAb did not. In the second experiment, inhibition of resolution of PCP in the anti-CD40L mAb group was not the result of blocking CD4+ T cell-dependent activation of PC-specific B cells. The results are consistent with the possibility that resistance to PCP may involve interaction between B cells and CD4+ T cells via the CD40-CD40L pathway. However, results additionally indicate that inhibition of CD40-CD40L interaction ablates resistance to PCP by inhibiting the interaction of T cells with some cell other than B cells.
Superantigen-induced collagenase gene expression in human IFN-gamma-treated fibroblast-like synoviocytes involves prostaglandin E2. Evidence for a role of cyclooxygenase-2 and cytosolic phospholipase A2.
Authors: Mehindate K , al-Daccak R , Dayer J M , Kennedy B P , Kris C , Borgeat P , Poubelle P E , Mourad W
Published: December 2022

MHC class II molecules expressed in lymphoid and nonlymphoid cells act as signal-transducer molecules. We demonstrate that engagement of MHC class II molecules on human IFN-gamma-treated fibroblast-like synoviocytes by their natural ligand, the staphylococcal enterotoxin A (SEA), selectively induces the production of interstitial collagenase over the expression of the tissue inhibitor of metalloproteinase (TIMP). Collagenase gene expression required de novo protein synthesis and was accompanied by high levels of PGE2 production, suggesting its implication in this response. Two inhibitors that affect prostaglandin biosynthesis, indomethacin and arachidonyl-trifluoromethyl-ketone, inhibited both PGE2 production and collagenase gene expression. The addition of exogenous PGE2 to inhibitor-treated cells partially restored the SEA-induced collagenase, indicating a role for PGE2 in this response. As cyclooxygenases (COX-1 and -2), cytosolic phospholipase A2 (cPLA2), and secreted PLA2 (sPLA2) are the enzymes potentially implicated in prostaglandin synthesis, their involvement in SEA-induced collagenase was investigated. The mRNA levels of COX-2 and cPLA2 rapidly increased following ligation of MHC class II molecules, while COX-1 and sPLA2 mRNA levels were unchanged and transiently depressed, respectively. SEA-induced COX-2 mRNA was translated adequately to protein, whereas cPLA2 protein level was not enhanced, but rapidly phosphorylated, a process previously linked to the enzyme activation. In conclusion, this work demonstrates a selective induction of collagenase gene expression over its natural inhibitor TIMP in human IFN-gamma-treated fibroblast-like synoviocytes mediated, at least in part, by PGE2, and provides evidence that signaling via MHC class II molecules induces the production of PGE2 through enhanced production of COX-2 and possibly activation of the cPLA2.
Molecular characterization of fusion regulatory protein-1 (FRP-1) that induces multinucleated giant cell formation of monocytes and HIV gp160-mediated cell fusion. FRP-1 and 4F2/CD98 are identical molecules.
Authors: Ohgimoto S , Tabata N , Suga S , Nishio M , Ohta H , Tsurudome M , Komada H , Kawano M , Watanabe N , Ito Y
Published: December 2022

Fusion regulatory protein (FRP)-1 regulates virus-mediated cell fusion and fusion of monocytes. Eleven of fifteen N-terminal amino acids of FRP-1 were the same as the amino acid sequence of 4F2/CD98 heavy chain. FRP-1 molecules were detected in Con A- or IL-2-stimulated lymphocytes, while FRP-1 was rare on resting lymphocytes. These properties of FRP-1 are similar to those of 4F2/CD98. Treatment of monocytes with anti-4F2/CD98 mAbs resulted in cell fusion, and other mAbs directed against 4F2/CD98 induced formation of multinucleated giant cells of Cd+U2ME-7 cells, a CD4+U937 cell line transfected with the HIV gp160 gene. Both anti-4F2/CD98 and anti-FRP-1 mAbs reacted with murine L929 cells expressing human 4F2/CD98 transiently or constitutively. When Newcastle disease virus (NDV)-infected L929 cells expressing human FRP-1/CD98 were incubated with mAb 4-5-1, an anti-FRP-1 mAb, multinucleated giant cells were induced; thus, FRP-1/CD98 molecules expressed in L929 cells are functional for fusion regulatory activity.
Two structurally distinct kappa B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages.
Authors: Ohmori Y , Fukumoto S , Hamilton T A
Published: December 2022

The mouse KC gene is an alpha-chemokine gene whose transcription is induced in mononuclear phagocytes by LPS. DNA sequences necessary for transcriptional control of KC by LPS were identified in the region flanking the transcription start site. Transient transfection analysis in macrophages using deletion mutants of a 1.5-kb sequence placed in front of the chloramphenicol acetyl transferase (CAT) gene identified an LPS-responsive region between residues -104 and +30. This region contained two kappa B sequence motifs. The first motif (position -70 to -59, kappa beta 1) is highly conserved in all three human GRO genes and in the mouse macrophage inflammatory protein-2 (MIP-2) gene. The second kappa B motif (position -89 to -78, kappa B2) was conserved only between the mouse and the rat KC genes. Consistent with previous reports, the highly conserved kappa B site (kappa B1) was essential for LPS inducibility. Surprisingly, the distal kappa B site (kappa B2) was also necessary for optimal response; mutation of either kappa B site markedly reduced sensitivity to LPS in RAW264.7 cells and to TNF-alpha in NIH 3T3 fibroblasts. Although both kappa B1 and kappa B2 sequences were able to bind members of the Rel homology family, including NF kappa B1 (P50), RelA (65), and c-Rel, the kappa B1 site bound these factors with higher affinity and functioned more effectively than the kappa B2 site in a heterologous promoter. These findings demonstrate that transcriptional control of the KC gene requires cooperation between two kappa B sites and is thus distinct from that of the three human GRO genes and the mouse MIP-2 gene.
Simultaneous cross-linking by two nontriggering bivalent ligands causes synergistic signaling of IgE Fc epsilon RI complexes.
Authors: Posner R G , Subramanian K , Goldstein B , Thomas J , Feder T , Holowka D , Baird B
Published: December 2022

We have used two bivalent ligands that bind IgE to study the relationship between the aggregation of receptors with high affinity for IgE (Fc epsilon RI) and the responses (receptor immobilization, Ca2+ influx, and degranulation) of rat basophilic leukemia (RBL-2H3) cells. One of these is a symmetric bivalent ligand, N,N'-bis[[epsilon-[(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L- cystine ((DCT)2-cys), which binds specifically to the combining sites of a mAb anti-DNP IgE and efficiently cross-links cell surface IgE, but does not trigger significant degranulation or increases in intracellular Ca2+. Several lines of evidence, including lateral mobility measurements, indicate that this ligand preferentially forms stable cyclic complexes containing two (DCT)2-cys and two IgE. The second ligand is a mAb anti-IgE, B1E3, which causes lateral mobility changes consistent with dimerized IgE-Fc epsilon RI and also does not trigger increases in intracellular Ca2+ or degranulation. The two ligands together trigger robust responses. In the presence of B1E3, (DCT)2-cys causes immobilization of IgE-Fc epsilon RI in a broad concentration range; in a more narrow concentration range, it is a potent stimulant of changes in both degranulation and Ca2+. We have compared the dose-response curves for cellular activation to simulated IgE aggregation curves, i.e., curves that predict the equilibrium IgE aggregate size distribution as a function of the (DCT)2-cys concentration. Our results indicate that maximal cellular activation occurs at a much higher (DCT)2-cys concentration than maximal IgE aggregation. When IgE aggregation is maximal, almost all aggregated IgE is in cyclic dimers. Thus, cyclic dimers appear to be functionally ineffective, even after they have been cross-linked by B1E3. Aggregated IgE-Fc epsilon RI that is effective in stimulating a cellular response may have particular structural or dynamic properties that allow critical interactions for initiating the signaling cascade.
Influence of heavy chain constant regions on antigen binding and HIV-1 neutralization by a human monoclonal antibody.
Authors: Cavacini L A , Emes C L , Power J , Desharnais F D , Duval M , Montefiori D , Posner M R
Published: December 2022

F105, a neutralizing IgG1 kappa human mAb, is reactive with a discontinuous epitope within the gp120 CD4 binding site. Because isotype usage may affect Ab function, we examined the effect of isotype on Ag/Ab interactions and HIV-1 neutralization. An IgG3 kappa Ab was prepared by linking the variable regions of F105 to cloned human kappa and gamma 3 constant regions. Immunoreactivity of F105 IgG1 and IgG3 with IIIB-, MN-, and RF-infected cells was equivalent. Inhibition of binding and fusion of IIIB to uninfected cells and neutralization of IIIB virus was comparable for F105 IgG1 and IgG3, with 14 to 23 micrograms/ml required for 90% neutralization. In contrast, F105 IgG3 was marginally more effective at inhibition of MN binding/fusion and significantly more effective at neutralization of MN virus (62 micrograms/ml for IgG3 and > 100 micrograms/ml for IgG1 to achieve 90% neutralization). Despite high affinity binding to RF-infected cells, F105 IgG1 minimally neutralizes free RF virus. F105 IgG3 is dramatically more effective against the RF isolate, with 2 to 20 micrograms/ml of Ab required for 50% neutralization. Both isotypes were relatively ineffective at inhibition of RF binding/fusion. Thus, whereas affinity with native Ags on the surface of HIV-1-infected cells was unaffected by heavy chain constant regions, Ab isotype can strongly influence virion neutralization. Structural changes in gp120, as a result of increased flexibility conferred by the elongated IgG3 hinge region, are suggested as a possible mechanism to increase neutralization of selected HIV-1 isolates. These results may have significant implications in the design of immunotherapeutic and vaccine agents.
Laser light suicide of proliferating virus-specific CD8+ T cells in an in vivo response.
Authors: Tripp R A , Lahti J M , Doherty P C
Published: December 2022

Virus-specific CD8+ CTL precursor (CTLp) frequencies are a function of rates of clonal expansion and loss, both of which are difficult to assess in vivo. Proliferating T cells incorporate the nucleoside analogue 5-bromo-2'-deoxyuridine (BrdU), making them sensitive to light-induced apoptotic cell death (suicide). Mice were infected with an influenza A virus, then given 50 mg of BrdU 24 h before sampling. Exposure of freshly isolated, CD8-labeled T cells to the laser beam of the flow cytometer resulted in the elimination of > 90% of the responding CTLp. The effect was obvious for the regional lymph node from day 6 to day 12 after priming, indicating continued cycling over a 7-day interval. Thus, as CTLp frequencies remain fairly constant from 7 to 30 days after infection, the persistent increase (> 30x) in numbers must be accompanied by a very substantial loss of virus-specific CD8+ T cells.
Rapid rejection of H2k and H2k/b bone marrow cell grafts by CD8+ T cells and NK cells in irradiated mice.
Authors: Davenport C , Kumar V , Bennett M
Published: December 2022

Acute rejection of transplanted bone marrow cell (BMC) grafts can occur within 48 h in unsensitized, lethally irradiated mice, and NK cells have been implicated as the effector cells. Recently, we observed that both CD8+ TCR-alpha beta+ T and NK cells of irradiated mice could rapidly reject allogeneic lymph node cell grafts. In this study, we evaluated the ability of NK and CD8+ T cells to mediate rejection of H2k or H2k/b BMC grafts by pretreating groups of mice with depleting mAbs. H2k BMC were transplanted into syngeneic, B6 (H2b), BALB/c (H2d), NZB (H2d), and (NZB x B6)F1 (NZB6F1, H2d/b) hosts. Proliferation measured 5 days after cell transfer indicated that syngeneic, B6, and BALB/c hosts accepted H2k BMC grafts. However, CD8+ T cells from NZB and poly I:C-treated BALB/c hosts, and NK cells from poly I:C-treated NZB6F1, hosts, rejected H2k BMC grafts. In NZB6F1 hosts, there was an added effect of anti-TCR-alpha beta and anti-NK1.1 mAbs. It is possible that T and NK cells cooperate in rejecting H2k or H2k/b BMC grafts in certain hosts. Transplantation of H2k/b, but not H2k/d, BMC into similar recipients had the same fate as H2k BMC. Thus, certain CD8+ T cells may share a similar recognition system with NK cells.
Effect of gene-targeted mutation in TNF receptor (p55) on contact hypersensitivity and ultraviolet B-induced immunosuppression.
Authors: Kondo S , Wang B , Fujisawa H , Shivji G M , Echtenacher B , Mak T W , Sauder D N
Published: December 2022

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic proinflammatory cytokine. TNF-alpha has been implicated in the pathogenesis of delayed-type hypersensitivity reactions such as allergic contact hypersensitivity and has been suggested as a mediator of ultraviolet B (UVB)-induced immunosuppression. Conflicting reports, however, exist concerning the effects of TNF-alpha on contact hypersensitivity (CHS). To determine the role of TNF-alpha in the generation and regulation of CHS, gene-targeted mutant mice lacking TNF-receptor (p55) gene (TNF-R1(-) mice) were treated with dinitrofluorobenzene (DNFB) to induce CHS. TNF-R1(-) mice showed significant hyperresponsiveness in CHS (152.8 +/- 20.9%, p < 0.025) compared with normal syngeneic mice (C57BL/6) assessed by ear swelling. To determine whether UVB can induce suppression in TNF-R1(-) mice, mice were irradiated on the shaved abdomen with 96 mj/cm2 UVB and 3 days later they were painted with 0.5% DNFB (sensitization dose), followed 5 days later with 0.2% DNFB to the left ear (challenge dose). Significant suppression of CHS was observed both locally (sensitization on irradiated site) and systemically (sensitization on unirradiated site) in UVB-irradiated TNF-R1(-) mice as well as in normal mice. To rule out possible signaling through p75 TNF-R, the mice were treated with anti-TNF-alpha Ab (V1q), which can neutralize any TNF effects through either receptor. V1q had no effect on these phenomena observed in TNF-R1(-) mice. These results suggest that TNF-alpha plays a regulatory role in CHS but is not required to induce UVB-mediated immunosuppression.
IFN-gamma receptor-deficient mice are hypersensitive to the anti-CD3-induced cytokine release syndrome and thymocyte apoptosis. Protective role of endogenous nitric oxide.
Authors: Matthys P , Froyen G , Verdot L , Huang S , Sobis H , Van Damme J , Vray B , Aguet M , Billiau A
Published: December 2022

Mice with a disruption of the IFN-gamma receptor alpha-chain gene (IFN-gamma R alpha o/o mice) were found to be significantly more sensitive than their wild-type counterparts to induction of the anti-CD3-induced disease syndrome. Specifically, when given a selected dose of anti-CD3 Ab, IFN-gamma R alpha o/o mice developed severe hypothermia and hypoglycemia, leading to 100% mortality within 72 h. In contrast, wild-type mice failed to develop overt pathologic manifestations and survived. Histologic examination revealed apoptosis in thymuses and spleens, which were significantly more pronounced in the mutant than in the wild-type mice, as confirmed by flow cytometric and DNA electrophoretic analysis. Apoptosis affected mainly CD4+CD8+ and CD4+CD8- thymocytes. Other histologic alterations were steatosis in livers, and erythrocyte extravasation and infiltration of apoptotic cells in lungs, all of which were exclusively observed in IFN-gamma R alpha o/o mice. Blood levels of TNF, IL-2, IL-6, and IL-10 were slightly more elevated in IFN-gamma R alpha o/o mice, but insufficiently so to explain increased disease severity. Thus, even more elevated cytokine levels in wild-type mice receiving high doses of anti-CD3 Ab were not associated with morbidity or apoptosis. Blood levels of IFN-gamma were barely detectable in anti-CD3-challenged wild-type mice, but were relatively high in the mutant mice. Increased susceptibility of IFN-gamma R alpha o/o mice was associated with impaired nitric oxide (NO) production, as indicated by significantly lower plasma nitrite levels and by more transient expression of spleen inducible NO synthase mRNA. Moreover, treatment of wild-type mice with the NO synthase inhibitor N-nitro-L-arginine methylester resulted in increased anti-CD3-induced morbidity and mortality. The data indicate that IFN-gamma R alpha o/o mice produce less NO and are therefore more sensitive than wild-type mice to the deleterious effect of anti-CD3 Ab.
Murine CD4+ T cells undergo TCR-activated adhesion to extracellular matrix proteins but not to nonantigenic MHC class II proteins.
Authors: O'Rourke A M , Lasam M C
Published: December 2022

Engagement of the TCR modulates the avidity of several receptors that play key roles in lymphocyte adhesion and/or signal transduction, including CD8, CD11a/CD18 (LFA-1), CD2, and several beta 1-integrins. Here, we investigated whether CD4+ T cells similarly undergo TCR-regulated adhesion to isolated MHC class II proteins through CD4. Strong adhesion of a number of CD4+ T cell clones to immobilized antigenic peptide/class II complexes was readily detectable. Adhesion to antigenic class II proteins was CD4 dependent and inhibited by pretreatment of T cells with the protein tyrosine kinase inhibitor herbimycin A, suggesting that adhesion requires TCR- and/or CD4-derived signal transduction. Treatment of T cells with anti-TCR Ab strongly increased subsequent adhesion to the extracellular matrix proteins, fibronectin and vitronectin, but, significantly, not to immobilized nonantigenic class II proteins. Suboptimal densities of antigenic peptide/class II complexes also activated adhesion of T cells to coimmobilized fibronectin or vitronectin, and this resulted in production of IFN-gamma to levels exceeding those stimulated by optimal densities of antigenic class II complexes alone. However, no augmentation of adhesion or cytokine secretion occurred when self or third party class II proteins were coimmobilized with antigenic class II complexes. The present results, therefore, suggest fundamental differences in the mechanism by which the TCR regulates coreceptor adhesion in CD4+ and CD8+ T cells.
Inhibition of T cell costimulation by VCAM-1 prevents murine graft-versus-host disease across minor histocompatibility barriers.
Authors: Schlegel P G , Vaysburd M , Chen Y , Butcher E C , Chao N J
Published: December 2022

Activation of T cells leading to graft-vs-host disease (GVHD) requires two signaling events: the Ag-specific signal generated through the engagement of the TCR/CD3 complex with antigenic peptide fragments presented by MHC molecules on APCs and the second signal provided through additional costimulatory ligands. T cells have preferential costimulatory requirements depending on their state of activation-induced maturation. In the present study, we investigated the role of the receptor-ligand pair VLA-4 (alpha 4 beta 1) and VCAM-1 in allogeneic T cell responses in vitro and in vivo. Anti-VCAM-1 mAb effectively inhibited mixed lymphocyte culture (MLC) across several major MHC barriers and secondary MLC across minor histocompatibility Ags (95.5% and 90.0% inhibition, respectively). In contrast, anti-VLA-4 mAb inhibited a CD8(+)-mediated primed CTL response in vitro by 100%, yet had little effect on proliferative responses. In the B10.D2/nSnJ-->BALB/c (both H-2d) system of GVHD, BALB/c received anti-VCAM-1, or anti-VLA-4 or controls NS-1 or Y13-259 for the first 5 wk after transplant. Anti-VLA-4 mAb delayed the onset of GVHD, but failed to reduce incidence, severity or GVHD-related mortality. In contrast, anti-VCAM-1 reduced the incidence of GVHD from 100% (18/18) in control animals to 53.3% (8/15) (p < 0.01) on day 70 post-transplant and significantly decreased GVHD-related mortality. Sixty percent (9/15) of anti-VCAM-1 recipients survived more than 180 days after transplant. Long-term engraftment of allogeneic bone marrow was documented in all transplanted mice by PCR analysis of a microsatellite region in the IL-1 beta gene.
Alpha and beta chemokines induce NK cell migration and enhance NK-mediated cytolysis.
Authors: Taub D D , Sayers T J , Carter C R , Ortaldo J R
Published: December 2022

Chemokines have been shown to play an important role in both the adhesion and migration of numerous leukocytic cell types, including granulocytes, monocytes, mast cells, and T lymphocytes. However, the biologic effects of chemokines on NK cells remain to be defined. Chemotaxis studies using purified human NK cells and a panel of human recombinant chemokines revealed that macrophage inflammatory protein (MIP)-1 alpha and IFN-inducible protein-10 (IP-10) are potent NK cell chemoattractants in vitro. Modest but significant chemotactic (not chemokinetic) responses were also observed in response to RANTES, MCP-1, MCP-2, MCP-3, and MIP-1 beta. Chemokine receptor expression on human NK cells was determined through displacement and Scatchard analyses, using a panel of radiolabeled chemokines, and revealed the presence of both distinct and shared chemokine receptors with affinities similar to those previously described for other cell types. Functional studies have also revealed that the beta chemokines and IP-10 are capable of augmenting NK- but not LAK- or ADCC-specific cytolytic responses in both a dose- and donor-dependent fashion. Neutralization analysis using Abs specific for various adhesion molecules revealed that NK:tumor cell conjugate formation is required for chemokine-induced NK killing. In addition, NK cells incubated in the presence of beta chemokines and IP-10 for 4 h induced the release of granule-derived serine esterases, suggesting a possible mechanism for chemokine-mediated NK killing. These results suggest that chemokines not only play an important role in the recruitment of NK cells, but also may be important mediators of NK cell degranulation augmenting local tumor cell destruction.
In vitro priming of tumor-reactive cytolytic T lymphocytes by combining IL-10 with B7-CD28 costimulation.
Authors: Yang G , Hellström K E , Mizuno M T , Chen L
Published: December 2022

Naive spleen cells from syngeneic mice generated tumor-reactive CTL following three cycles of in vitro culturing with IL-10 and cells from the P815 mouse mastocytoma that expressed the B7-1 or B7-2 costimulator. Unpurified as well as CD8-enriched naive splenocytes could be used for priming. The in vitro primed CTL were CD8+, and their recognition was MHC class I restricted. Both IL-10 and B7-transfected P815 cells were required for the priming. However, a combination of exogenous IL-10 and IL-2 in the presence of B7-negative wild-type P815 cells also induced tumor-reactive CTL. Injection of the neutralizing anti-IL-10 mAb JES-2A5 into mice reduced their ability to mount a primary CTL response after immunization with B7-1+ P815 cells, and inclusion of this mAb in the in vitro cultures inhibited a secondary CTL response. Adoptive transfer of the in vitro primed CTL had a therapeutic effect in mice with P815 established as an ascites tumor. Our results underscore an important role of IL-10 in the induction of a tumor-specific CTL response.
N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding.
Authors: Liao H X , Lee D M , Levesque M C , Haynes B F
Published: December 2022

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.
Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans.
Authors: Sztein M B , Tanner M K , Polotsky Y , Orenstein J M , Levine M M
Published: December 2022

Not only viruses, but certain parasites and bacteria as well, can elicit CTL involved in mediating protection. It has been surmised that CTL able to lyse Salmonella typhi-infected cells are likely to be important in protecting against S. typhi, an intracellular bacterial infection, but heretofore this has not been demonstrated. Consequently, the presence of CTL activity against S. typhi-infected cells was investigated in human volunteers immunized with attenuated vaccine strains of S. typhi. Oral immunization with S. typhi strain CVD 908 elicited circulating CTL effector cells capable of killing S. typhi-infected autologous EBV-transformed cells. CTL activity was observed after 6 to 8 days of in vitro expansion in the presence of S. typhi-infected autologous EBV-transformed cells. Maximum CTL activity was observed 29 days after immunization. Depletion of CD8+ T cells eliminated or markedly reduced the CTL activity, while depletion of CD4+ T cells did not affect CTL responses. CTL activity was blocked by mAbs to human class I MHC Ags, but not by mAbs to class II MHC Ags. This first demonstration that oral immunization of volunteers with attenuated S. typhi elicits CD8+ T cell, MHC class I-restricted, CTL responses raises the possibility that CTL activity might play a significant role in protection during typhoid fever. It also encourages the future use of such attenuated strains as liver vector vaccines to stimulate specific CTL against relevant foreign Ags.
Lipopolysaccharide stimulates the tyrosine phosphorylation of mitogen-activated protein kinases p44, p42, and p41 in vascular endothelial cells in a soluble CD14-dependent manner. Role of protein tyrosine phosphorylation in lipopolysaccharide-induced stimulation of endothelial cells.
Authors: Arditi M , Zhou J , Torres M , Durden D L , Stins M , Kim K S
Published: December 2022

Vascular endothelial cell (EC) injury or activation by LPS plays a critical role in the pathogenesis of Gram-negative meningitis and endotoxic shock. EC do not express membrane CD14, but respond to LPS in a soluble CD14-dependent manner. The signal transduction mechanisms involved in LPS-induced EC responses are largely unknown. We used bovine and human brain microvessel EC (BBMEC, and HBMEC) to study LPS-induced protein tyrosine phosphorylation. LPS rapidly induced the tyrosine phosphorylation of several proteins in BBMEC and HBMEC, which was detectable by 5 to 15 min, reached a maximum by 30 min, and declined by 60 to 90 min. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 0.1 ng/ml and was dose dependent up to 100 ng/ml. Similar changes in tyrosine phosphorylation were induced by smooth and rough LPS as well as lipid A, but not by the inactive lipid A analogue, Rhodopseudomonas sphaeroides diphosphoryl lipid A. Pretreatment of EC with the tyrosine kinase inhibitor, herbimycin A, inhibited LPS-stimulated protein tyrosine phosphorylation and LPS-mediated lactic dehydrogenase release from BBMEC and IL-6 release from HBMEC in a dose-dependent manner. Three proteins with apparent m.w. of 44, 42, and 41 kDa were predominant among the LPS-induced tyrosine phosphoproteins, and they were identified as mitogen-activated protein kinase isoforms ERK1, ERK2, and p38, respectively. LPS-induced protein tyrosine phosphorylation in HBMEC and BBMEC was soluble CD14 dependent, since pretreatment of these cells with anti-hCD14 mAb inhibited the LPS-induced tyrosine phosphorylation of p44, p42, and p41. Additionally, LPS induced a mobility shift in p44 and p42 mitogen-activated protein kinase isozymes, which was inhibited by herbimycin A pretreatment of the EC. These findings demonstrate for the first time that increased protein tyrosine phosphorylation and activation of mitogen-activated protein kinases occur rapidly after LPS stimulation of EC in the presence of soluble CD14. Our data also suggest that a herbimycin-sensitive step, presumably a tyrosine kinase, is involved in mediating LPS-induced human EC activation and IL-6 secretion.