bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of HLA-DQ binding properties by differences in class II dimer stability and pH-dependent peptide interactions.
Authors: Buckner J , Kwok W W , Nepom B , Nepom G T
Published: December 2022

Intermolecular interactions between peptides and class II molecules intracellularly are likely to be influenced by different pH conditions in different endosomal compartments. We compared the pH-dependent binding of peptides to HLA-DQ alleles 3.1 and 3.2, two well-studied human class II molecules with limited structural variability but different genetic associations with autoimmune diseases. Binding of the "promiscuous" binding peptide 34P3A as well as that of several allele-specific peptides were optimal for DQ3.2 at relatively acidic pH, compared with DQ3.1, with a pH optimum for DQ3.2 at pH 5.5 and for DQ3.1 at pH 6.5. A specific polymorphism at residue 57 of the DQ molecule accounted for some, but not all, of this difference. When the intrinsic stability of the class II dimer was assessed using partially denaturing gel electrophoresis after incubation at different pH, the DQ3.2 molecule demonstrated relative instability at neutral pH compared with DQ3.1, again suggesting a preference for peptide binding at relatively acidic conditions for HLA-DQ3.2. Interestingly, this preference for binding at acidic pH was not found for a class II-associated invariant chain peptide. Intrinsic alphabeta dimer stability and the pH optima for peptide binding studied in this report are likely to be significant determinants for allelic differences in DQ binding profiles in vivo.
Altered orientation of streptococcal superantigen (SSA) on HLA-DR1 allows unconventional regions to contribute to SSA Vbeta specificity.
Authors: Stevens K R , Van M , Lamphear J G , Rich R R
Published: December 2022

Crystallographic studies reveal even distantly related bacterial superantigens (SAg) to adopt a common structural topology. Mutational analyses confirm that this shared folding pattern often confers a conserved function to analogous residues in different SAg, albeit with specificities for particular TCR or MHC class II molecules. It was thus surprising that the streptococcal SAg SSA differed from related SAg in the location of its Vbeta-determining residues. Because it seemed unlikely that SSA would deviate significantly from an SAg-like topology, we hypothesized that variations in SSA Vbeta-determining regions might result from differences in SSA-MHC class II interactions relative to other SAg during SSA presentation to the TCR. Comparison of the DR1-binding properties of SSA with its closest homologue SEB found different amino acid positions within SAg primary sequences to contribute to SSA-DR1 and SEB-DR1 interactions, and suggested that SSA bound DR1 with an altered orientation relative to SEB. The common involvement of DR1 alpha39K, however, predicted that the two SAg bound overlapping sites on DR1. Nevertheless, SSA and SEB did not effectively cross-compete for DR1 binding and had opposite patterns of DR1-binding affinity in the presence of distinct DR1-expressing cell lines. The data thus suggest that SSA and SEB bind not only with different orientations on DR1, but may bind preferentially to distinct DR1 subsets delineated by cell-specific factors. Differences in orientation of SSA on DR1 and/or interaction of SSA with particular DR1 subsets may explain why unconventional regions influence SSA TCR Vbeta specificity.
The roles of CD11/CD18 and ICAM-1 in acute Pseudomonas aeruginosa-induced pneumonia in mice.
Authors: Qin L , Quinlan W M , Doyle N A , Graham L , Sligh J E , Takei F , Beaudet A L , Doerschuk C M
Published: December 2022

Neutrophil accumulation in response to Pseudomonas aeruginosa in the lungs is mediated through CD11/CD18. This study determined the roles of CD11a, CD11b, and intercellular adhesion molecule (ICAM)-1 in P. aeruginosa-induced pneumonia and compared the function of ICAM-1 using Abs or ICAM-1 mutant mice. Anesthetized BALB/c mice pretreated with either Abs against CD11a, CD11b, ICAM-1, or rat IgG received intratracheal instillation of P. aeruginosa for 4 h. In other studies, ICAM-1 mutant and wild-type mice received either anti-ICAM-1 Ab or rat IgG followed by instillation of P. aeruginosa. The data show that Abs against CD11a, CD11b, and ICAM-1 in BALB/c mice inhibited neutrophil emigration by 79, 81, and 56%, respectively. ICAM-1 mutant mice showed no inhibition of neutrophil emigration compared with wild-type mice. Pretreatment with anti-ICAM-1 Ab inhibited neutrophil emigration in wild-type (129/SvxC57) mice by 67% but had no effect in ICAM-1 mutant mice, suggesting that the Ab was acting specifically through recognition of its Ag. We conclude that CD11a and CD11b are required for neutrophil emigration. The observed function of ICAM-1 varies depending on the method by which it is inhibited. Abs may overestimate function by altering other cellular functions or mutant mice may develop alternative pathways of emigration.
A differential role for cell shape in neutrophil tethering and rolling on endothelial selectins under flow.
Authors: Finger E B , Bruehl R E , Bainton D F , Springer T A
Published: December 2022

We investigated the role of neutrophil microvilli in interactions with E-selectin and P-selectin in hydrodynamic shear flow by disruption with cytochalasin B, hypotonic swelling, and chilling. Cytochalasin B only marginally reduced microvilli numbers (from 30 +/- 6 to 16 +/- 6 per cell perimeter, p < 0.005) as shown by electron microscopy, completely disrupted tethering in shear flow to E-selectin and P-selectin, increased the strength of rolling adhesions on E-selectin and P-selectin, and increased cell deformability in shear flow with a likely increase in the area of cell:substrate contact. Hypoosmotic swelling markedly reduced microvilli number (to 6 +/- 5 per perimeter, p < 0.005), almost completely inhibited tethering on E- and P-selectin, and increased the strength of rolling adhesions on P-selectin but not on E-selectin. Chilling almost completely abolished microvilli (to 3 +/- 3 per perimeter, p < 0.005), but pseudopod-like structures were present, and had little effect on tethering in flow. Immunogold labeling of L-selectin, which is normally clustered on tips of microvilli, showed that in the absence of microvilli it remained in small clusters. Our studies show that alterations in cell morphology and viscoelasticity can have opposing effects on tethering and rolling, showing that they are independently regulatable. Furthermore, our results suggest that the association of molecules that mediate rolling with microvilli tips may be important not just to enhance presentation, but for other functions such as to promote resistance to extraction from the membrane or cooperative interactions among clustered receptors.
Cross-linking of intercellular adhesion molecule 1 (CD54) induces AP-1 activation and IL-1beta transcription.
Authors: Koyama Y , Tanaka Y , Saito K , Abe M , Nakatsuka K , Morimoto I , Auron P E , Eto S
Published: December 2022

Leukocytes adhere to target cells through their integrins and play a crucial role in self-defense, inflammation, and differentiation. Intercellular adhesion molecule-1 (ICAM-1; CD54) is a representative ligand for integrins and is expressed on many cell types, some of which are targets for leukocyte adhesion. Recent studies suggest that adhesion molecules function not only as a cellular glue, but also as a signal transducer. However, it remains to be clearly defined whether engagement of ICAM-1 is able to induce activation signals in target cells. In rheumatoid synovium, synovial cells are known to express abundant ICAM-1 and produce multiple inflammatory cytokines, such as IL-1beta. In this study, we provide the first evidence that ICAM-1 engagement induces activation of the transcription factor AP-1 and transcription of the IL-1beta gene using a specific Ab to cross-link ICAM-1 on a rheumatoid synovial cell line (E11 cells). This evidence includes ICAM-1 cross-linking-dependent induction of 1) in situ IL-1beta transcription and protein synthesis, 2) transiently transfected chloramphenicol acetyltransferase (CAT) reporter plasmids containing both the IL-1beta LPS-responsive enhancer (between -3134 and -2729) as well as multiple copies of an AP-1 site from this enhancer (between -3117 and -3111), and 3) the binding of a Jun/Fos family complex to this AP-1 site. Thus, ICAM-1 not only functions as a glue for integrin binding, but also as a transducer for AP-1 activation signals important for IL-1beta gene transcription.
Chemical characteristics of Staphylococcus aureus molecules that have CD14-dependent cell-stimulating activity.
Authors: Kusunoki T , Wright S D
Published: December 2022

We have previously shown that Staphylococcus aureus contains molecules that bind CD14 and induce cellular responses. We have partially purified this activity and named the product active fraction (AF). Here, we further explore the chemical characteristics of AF. We show that 1) the activity is sensitive to base and hydrofluoric acid hydrolysis; 2) AF binds to Mono Q, indicating that it is negatively charged; 3) most activity in AF runs as a large species in aqueous buffer, but a small proportion runs with a size of <15 kDa by gel chromatography; and 4) native PAGE of AF yields a band of high apparent m.w., and SDS-PAGE yields a ladder of bands of lower apparent m.w. These data suggest that AF is a mixture of heterogeneous amphiphilic molecules that form tight micelles. These characteristics are shared by LPS, suggesting that AF has both functional and structural similarity to LPS. S. aureus cell wall has been shown to stimulate cells in a CD14-dependent fashion, and peptidoglycan (PGN) has thus been described as a ligand for membrane CD14. However, we found that the ability of S. aureus cell wall to stimulate CD14-bearing monocytes was enhanced by preincubation with soluble CD14 (sCD14). Upon treatment with sCD14, the cell-stimulating activity appeared predominantly in the supernatant, not in the insoluble PGN pellet. These findings are not consistent with a direct interaction of membrane CD14 with insoluble PGN. They suggest, rather, that the activity of cell wall may be caused by a sCD14-binding molecule that associates noncovalently with PGN.
Differential effects of macrophage- and granulocyte-macrophage colony-stimulating factors on cytokine gene expression during rat alveolar macrophage differentiation into multinucleated giant cells (MGC): role for IL-6 in type 2 MGC formation.
Authors: Lemaire I , Yang H , Lafont V , Dornand J , Commes T , Cantin M F
Published: December 2022

Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage (GM)-CSF stimulate the differentiation of rat alveolar macrophages (AM) into multinucleated giant cells (MGC) with distinct phenotypes (type 1 and type 2 MGC). In the present study, we analyzed the profile of cytokine gene expression induced respectively, by M-CSF and GM-CSF during rat AM differentiation using reverse transcription-PCR. Enhanced mRNA expression for IL-1alpha, TNF-alpha, and TGF-beta1 was observed 3 h after treatment with M-CSF (50 U/ml) or GM-CSF (50 U/ml). In contrast, IL-6 mRNA expression was increased by GM-CSF but not M-CSF. Kinetic analysis indicated that GM-CSF stimulated IL-6 expression early (1.5 h), with maximal effect observed at 24 h and up to 5 days thereafter. Increased mRNA levels for IL-6 were associated with higher IL-6 activity in the culture media of differentiating AM. IL-6 activity was stimulated 3 h after treatment with GM-CSF and increased with time (up to 5 days). Interestingly, addition of exogenous IL-6 (20-100 ng/ml) alone or in combination with GM-CSF to AM cultures increased slightly and selectively the formation of MGC with type 2 phenotype. Conversely, neutralization of endogenous IL-6 during AM differentiation into MGC inhibited significantly (up to 50%) the formation of type 2 MGC. These results suggest a role for IL-6 in the formation of type 2 MGC and provide some insights into the mechanisms of MGC formation and the processes that regulate it positively.
Calcium ionophore-induced secretion from mast cells correlates with myosin light chain phosphorylation by protein kinase C.
Authors: Ludowyke R I , Scurr L L , McNally C M
Published: December 2022

Rat basophilic leukemia mast cells (RBL-2H3) secrete histamine when activated by Ag. This secretion correlates with increased phosphorylation of myosin light chain by protein kinase C (PKC). Calcium ionophores (A23187) also elicit secretion, which is enhanced by PMA. To analyze the roles of Ca2+ and PKC in the secretory process, A23187-induced myosin light chain phosphorylation was examined in the presence and absence of PMA. A23187-induced secretion correlated best with myosin light chain phosphorylation by PKC, not with phosphorylation by myosin light chain kinase (MLCK). A23187 induced the translocation to membranes of the alpha, beta, delta, and epsilon isozymes of PKC. PMA not only increased the phosphorylation of myosin light chains at PKC-specific sites (Ser1 and Ser2) but also at sites attributed to MLCK (Ser19 and Thr18-Ser19). A23187 plus PMA induced higher levels of secretion concomitantly with increased myosin light chain phosphorylation at the PKC-specific sites. However, there was little correlation between the translocation of specific PKC isozymes and the phosphorylation of myosin light chains by PKC. Activation induced a novel triphosphorylated form of myosin light chain with a higher level of phosphorylation at the diphosphorylated MLCK sites. Quantitation of A23187 plus PMA-induced myosin light chain phosphorylation revealed that phosphorylation at PKC sites increased from zero to 0.35 mol/mol, was little changed at the monophosphorylated MLCK site (0.30 mol/mol), and increased from zero to 0.06 mol/mol at the diphosphorylated MLCK sites. Therefore, Ca2+-induced secretion correlates best with myosin light chain phosphorylation by PKC, and diphosphorylation by MLCK is unlikely to contribute to secretion.
Recruitment of lymphoblasts derived from peripheral and intestinal lymph to synovium and other tissues in normal rats and rats with adjuvant arthritis.
Authors: Spargo L D , Hawkes J S , Cleland L G , Mayrhofer G
Published: December 2022

Recruitment of [125I]iododeoxyuridine-labeled syngeneic lymphoblasts from thoracic duct (TD) lymph into periarticular tissues has been examined after intravenous administration to normal rats and to rats with adjuvant-induced arthritis. Uptake of label was observed in the inflamed paws of arthritic rats and cells were located in synovium and periarticular bone marrow by autoradiography. Uptake was greater with lymphoblasts from donors in the late prodromal phase of adjuvant-induced arthritis (arthritic donors) than from normal donors. With arthritic donors, recruitment of lymphoblasts from TD lymph was greater than from mesenteric duct lymph, suggesting that most of the joint-seeking lymphoblasts in arthritic rats arose in peripheral lymphoid tissues. Lymphoblasts from arthritic donors were also detected in the synovium of paws from normal rats. Recovery of lymphoblasts was monitored in other tissues; this revealed, in arthritic recipients, competition among extra-articular sites of inflammation (adjuvant injection site, draining lymph nodes, and lymph nodes draining affected joints), the lungs, and the inflamed synovium for recruitment of lymphoblasts from arthritic donors. In contrast, while some lymphoblasts from normal donors were recruited to inflamed joints, the small intestine was the main site of recruitment. The results reflect the known propensity of T lymphoblasts generated in peripheral lymphoid tissues to enter inflamed tissues. However, some mesenteric duct lymphoblasts also entered inflamed synovium. The observed pattern of recruitment of lymphoblasts to synovium is pertinent to the pathogenesis of arthritis, the potential roles of arthritogenic and "bystander" lymphocytes and the known links between the joints and inflammation in the intestine.
CD100 is associated with CD45 at the surface of human T lymphocytes. Role in T cell homotypic adhesion.
Authors: Herold C , Elhabazi A , Bismuth G , Bensussan A , Boumsell L
Published: December 2022

CD100 is a 150-kDa human surface glycoprotein implicated in T cell activation. Using BB18 and BD16 mAbs to two discrete epitopes of the CD100 molecule, we have shown previously that triggering the CD100 molecule through the BB18 epitope is comitogenic with PMA, whereas it lacks a detectable effect on CD2- and CD3-induced PBMC proliferation. Conversely, triggering the CD100 molecule through the BD16-defined epitope only exerts an effect on CD2- and CD3-induced PBMC proliferation. In the present study, we investigated the molecular relationship between CD100 and another surface structure involved in human T cell proliferation, the CD45 phosphatase. We show in this study that CD100 is associated with CD45 and that this association has functional significance. The association was demonstrated using coimmunoprecipitation and detection of CD45 enzymatic PTPase activity. Furthermore, we show that the association is increased during T cell activation and that triggering CD45 molecules through discrete epitopes induces the down-modulation of CD100 molecules at the cell surface. Interestingly, we demonstrate that this modulation could be attributed to the shedding of a soluble form of CD100 in the culture supernatant. Finally, one of the functional consequences of this T cell activation-induced CD100-CD45 association is revealed by the finding that CD100 mAbs have an effect on CD45-induced T cell aggregation.
delta opioid receptor expression is induced by concanavalin A in CD4+ T cells.
Authors: Miller B
Published: December 2022

Expression of delta opioid receptor mRNA in CD4+ T cells purified from Con A-stimulated murine splenocytes was demonstrated by reverse transcription-PCR and sequencing of the PCR products. mu and kappa opioid receptor mRNAs were not detected. Similar results were obtained in a single isolation of CD8+ T cells from Con A-stimulated splenocytes. mRNA for the delta opioid receptor was not detected in CD4+ T cells purified from either freshly isolated splenocytes or splenocytes cultured without Con A, and incubation of purified CD4+ T cells with Con A did not induce delta opioid receptor mRNA. It appears that quiescent CD4+ T cells do not express classic opioid receptors, but the delta-type receptor can be induced upon activation.
Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1.
Authors: Shier P , Otulakowski G , Ngo K , Panakos J , Chourmouzis E , Christjansen L , Lau C Y , Fung-Leung W P
Published: December 2022

We have generated mice deficient in the beta2 integrin LFA-1 by targeted disruption of the CD11a gene in embryonic stem cells. In vitro LFA-1 -/- cells exhibit a delayed proliferative response toward alloantigens in the MLR. In vivo the host-vs-graft reaction toward injected allogeneic cells is also reduced. Alloantigen-specific CTLs generated from LFA-1 -/- mice are impaired in their cytotoxic activity toward allogeneic spleen cells as well as cell line targets. The proliferative response of LFA-1 -/- splenocytes following stimulation by LPS, PMA plus ionomycin, or immobilized anti-CD3epsilon mAb is normal, but Con A-stimulated proliferation is greatly diminished. We observe typical edema formation in a delayed type hypersensitivity reaction to SRBC with normal extravasation of leukocytes and demonstrate recruitment of neutrophils to an LPS-induced inflammatory site in these mice, suggesting that LFA-1 does not play an essential role in lymphocyte homing and leukocyte extravasation. We further show that LFA-1 -/- mice are susceptible to metastasis of B16 melanoma tumors, although their in vitro NK cell activity appears normal. A study of LFA-1 -/- mice expressing transgenic TCRs indicates that thymic maturation and selection of T cells are unaffected by the loss of LFA-1. Our results indicate that LFA-1 is important for alloantigen-triggered T cell proliferation and cytotoxicity, for Con A stimulation of T cells, and in tumor rejection. It does not appear to play an essential role in lymphocyte homing and leukocyte extravasation or in T cell maturation and selection in the thymus.
Differential activation of human platelets induced by Fc gamma receptor II cross-linking and by anti-CD9 monoclonal antibody.
Authors: Qi R , Ozaki Y , Kuroda K , Asazuma N , Yatomi Y , Satoh K , Nomura S , Kume S
Published: December 2022

Platelet activation induced by anti-CD9 mAb, which depends upon Fc gammaRII, has been considered to be similar to that induced by Fc gammaRII cross-linking. In this work, we present several lines of evidence to suggest that the mode of platelet activation induced by anti-CD9 mAb is distinct from that induced by Fc gammaRII cross-linking. Ca2+ release from intracellular Ca2+ stores induced by anti-CD9 mAb depended almost totally upon thromboxane A2 production and released ADP, whereas that induced by Fc gammaRII was affected only minimally by these factors. Fc gammaRII cross-linking induced Ca2+ channel opening, which is dependent upon the depletion of intracellular Ca2+ stores. In contrast, anti-CD9 mAb appeared to directly open Ca2+ channels, irrespective of intracellular Ca2+ stores (Kuroda et al., 1995. J. Immunol. 155: 4427). The Ca2+ requirement for the Ca2+ channels opened by Fc gammaRII cross-linking was also distinct from that induced by anti-CD9 mAb. The early phase of Fc gammaRII tyrosine phosphorylation was dependent upon thromboxane A2 production with anti-CD9 mAb-induced activation, whereas that of Fc gammaRII cross-linking was not. p72(syk) and p53/56(lyn) appeared to associate with Fc gammaRII in platelet activation induced by Fc gammaRII cross-linking, whereas there was little, if any, association between Fc gammaRII and these tyrosine kinases in anti-CD9 mAb-induced activation. Piceatannol, a selective inhibitor of p72(syk), enhanced Fc gammaRII tyrosine phosphorylation induced by Fc gammaRII cross-linking, whereas it attenuated the process in anti-CD9 mAb-induced platelet activation. It is suggested that the regulatory mechanism of Fc gammaRII tyrosine phosphorylation differs between these two modes of platelet activation.
The length of treatment determines whether IFN-beta prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats.
Authors: Ruuls S R , de Labie M C , Weber K S , Botman C A , Groenestein R J , Dijkstra C D , Olsson T , van der Meide P H
Published: December 2022

The mechanism of action underlying the beneficial effect of IFN-beta in multiple sclerosis (MS) is not understood. To date, little information is available on the effects of IFN-beta in experimental autoimmune encephalomyelitis (EAE), the animal correlate of the human disease MS. Therefore, we investigated the effects of recombinant rat IFN-beta (rrIFN-beta) on EAE in Lewis rats with emphasis on a treatment regimen during the paralytic phase of the disease. The results indicated that rrIFN-beta dose-dependently inhibited disease activity with complete prevention at a s.c. dose of 300,000 U/day, provided that treatment was continued for 3 wk. Discontinuation of treatment on day 17 postimmunization resulted in a protracted and relapsing disease course with strongly enhanced clinical severity. Detailed immunohistology of central nervous system (CNS) tissue of protected animals revealed an almost complete absence of CNS lesions and a >90% reduction in the number of infiltrating leukocytes. Accordingly, isolation of mononuclear cells from spinal cord tissue of successfully treated EAE rats revealed a reduction of approximately 95% in the number of cells that produce IFN-gamma in response to the encephalitogenic peptide MBP63-88. Furthermore, rrIFN-beta significantly enhanced serum corticosterone levels, which showed an inverse relationship with disease activity. We show that rrIFN-beta can have both beneficial and detrimental effects on disease activity dependent on the timing and the duration of treatment. Beneficial effects on EAE are associated with inhibition of the extravasation of blood-derived mononuclear cells in the CNS.
The anti-beta2-glycoprotein I activity in human anti-phospholipid syndrome sera is due to monoreactive low-affinity autoantibodies directed to epitopes located on native beta2-glycoprotein I and preserved during species' evolution.
Authors: Tincani A , Spatola L , Prati E , Allegri F , Ferremi P , Cattaneo R , Meroni P , Balestrieri G
Published: December 2022

To characterize the reactivity pattern of Abs directed to beta2-glycoprotein I (anti-beta2GPI) in patients with anti-phospholipid syndrome, we have purified anti-beta2GPI Abs by affinity chromatography using the IgG fractions from sera of five different anti-phospholipid syndrome patients. Affinity-purified anti-beta2GPI were shown to be representative of Abs found in human sera because their activity could be virtually abolished from the IgG preparations after repeated absorptions on immobilized human beta2GPI column. Our results show that affinity-purified anti-beta2GPI: 1) do react with beta2GPI in the absence of any phospholipid, as demonstrated by the lack of phosphorus contaminant in the employed reagents, as well as by their comparable binding activity before and after extensive delipidation procedure; 2) can recognize beta2GPI regardless of its origin from different animal species; 3) are able to bind soluble beta2GPI with a mean Kd value of 4.65 x 10(-6) M (range 3, 4-7, 2 x 10(-6) M); 4) significantly enhance their binding avidity when beta2GPI is linked to a solid support; and 5) appear to be mainly monoreactive autoantibodies. In conclusion, we have shown that human polyclonal anti-beta2GPI are low affinity, mainly monoreactive autoantibodies directed to an epitope located on native beta2GPI, preserved along the species evolution.
Prolactin administration following hemorrhagic shock improves macrophage cytokine release capacity and decreases mortality from subsequent sepsis.
Authors: Zellweger R , Zhu X H , Wichmann M W , Ayala A , DeMaso C M , Chaudry I H
Published: December 2022

Although prolactin is reported to counteract the immunosuppressive effects of glucocorticoids, cyclosporine, and morphine, it remains unknown whether prolactin has any salutary effects on the depressed immune responses following severe hemorrhage. To study this, mice were bled to and maintained at a mean arterial pressure of 35 mm Hg for 60 min, then adequately resuscitated and divided into two groups. One group received saline vehicle, while the other group received prolactin (100 micro g/25 g body weight, s.c.) immediately before resuscitation. Two hours thereafter, peritoneal (pMphi) and splenic macrophages (sMphi) were harvested and assessed not only for their ability to release IL-1 and IL-6, but also for cytokine gene expression using semiquantitative reverse transcription and PCR. In an additional group, mice were subjected to sepsis by cecal ligation and puncture 3 days after hemorrhage. Hemorrhage markedly decreased the ability of pMphi and sMphi to release IL-1 and IL-6. This was, however, associated with increased mRNA expression for IL-1beta and IL-6 and increased serum corticosterone levels. Following prolactin treatment of hemorrhaged mice, IL-1beta and IL-6 mRNA levels as well as cytokine release capacity and blood corticosterone levels were comparable to the values in sham animals. Prolactin also improved the survival of animals subjected to sepsis after hemorrhage. Thus, the immunosuppression following hemorrhage appears to be mediated and modulated by hormones from the hypothalamic-pituitary-adrenal axis. Furthermore, prolactin represents a novel immunomodulating hormone for the treatment of immunodepression encountered after hemorrhagic shock and for decreasing the mortality from subsequent sepsis under those conditions.
Specific inhibition of T lymphocyte coactivation by triggering integrin beta 1 reveals convergence of beta 1, beta 2, and beta 7 signaling pathways.
Authors: Woodside D G , Teague T K , McIntyre B W
Published: December 2022

T cell coactivation is a dynamic process subject to integrin-dependent positive and negative regulation. Costimulation of human peripheral blood T cells by CD3 mAb OKT3 in conjunction with anti-alpha 4 has been shown to be down-regulated by the anti-beta 1.1 epitope-specific mAb 18D3. As expected, maximal costimulation induced by alpha 4-specific mAb L25 was inhibited (70%) by the addition of soluble mAb 18D3. Surprisingly, soluble mAb 18D3 inhibited maximal proliferation induced by the costimulatory alpha 4 beta 7-specific mAb ACT-1 by 40%, thus demonstrating that one integrin subfamily can regulate the activity of another. To determine whether mAb 18D3 could regulate more than alpha 4-associated integrin-mediated costimulation, non-alpha 4 integrins were tested. mAb 18D3 inhibited maximal proliferation induced by alpha 4-specific mAb 3D6, and an alpha 4-specific mAb 16. This clearly demonstrates that a variety of integrin costimulatory molecules (of the beta 1, beta 2, and beta 7 subfamilies) can be regulated negatively by mAb 18D3. To analyze the specificity of this negative regulation, other cell surface costimulatory molecules were tested for susceptibility to mAb 18D3. Although Abs specific for CD4, CD26, CD28, CD44, CD45RA, or CD45RO were sufficient to activate T cells when co-immobilized with anti-CD3 mAb, all were refractory to the inhibitory effects of mAb 18D3. Inhibition of T cell activation directly correlated with diminished IL-2 production. This suggests that mAb 18D3 selectively regulates integrin-dependent T cell activation by delivering a negative effect at some common point utilized by various integrin subfamilies.
Molecular mimicry between an immunodominant amino acid motif on the 47-kDa lipoprotein of Treponema pallidum (Tpp47) and multiple repeats of analogous sequences in fibronectin.
Authors: Baughn R E , Jiang A , Abraham R , Ottmers V , Musher D M
Published: December 2022

Molecular mimicry, resulting from structural similarities between self-determinants on host Ags and an organism's antigenic determinants (epitopes), can incite autoimmune events in certain bacterial and viral diseases. In the course of comprehensively mapping the 47-kDa lipoprotein (Tpp47) of Treponema pallidum subsp. pallidum using an overlapping synthetic peptide strategy, we identified a major immunoreactive epitope (411PGTEYT416) that exhibited considerable motif identity with multiple repeats of analogous linear sequences found in mammalian fibronectins. To further explore the importance of this motif as a probable instigator in the induction of polyspecific cross-reactive Abs, mimetic variants were synthesized for immunologic studies. Mimetics with ala (A) replacements in each amino acid position were used to determine which residues were critical for Ab binding. Animals immunized with two mimetics (PGTEYT or PGSEYT) coupled to tetanus toxoid exhibited: 1) modified responses when challenged with viable T. pallidum; and 2) classical Arthus reactions when challenged intradermally with either motif linked to a different carrier. The cross-reactive nature of the Ab responses to both mimetics was confirmed in a variety of ELISAs using mimetics, fibronectins, and collagens. Inhibition-ELISA studies with both fibronectin and an unrelated mimetic of the RGD motif suggest that intra- and intermolecular epitope spreading occurs following mimetic immunization and involves additional self-epitopes. These observations suggest that although molecular mimicry plays a pivotal role in initially triggering the anti-fibronectin and anti-collagen responses associated with disseminated syphilis, expansion of those autoimmune responses may be due to other self-epitopes once tolerance is abrogated.
CTL recognition of an altered peptide associated with asparagine bond rearrangement. Implications for immunity and vaccine design.
Authors: Chen W , Ede N J , Jackson D C , McCluskey J , Purcell A W
Published: December 2022

The extent to which peptides containing chemically and post-translationally modified amino acid side chains are recognized by primed CTL has not been clearly defined. We report on the CTL recognition of a MHC class I-restricted peptide containing a cyclized asparagine (succinimide) residue. This modification of the asparagine side chain is a common intermediate structure during deamidation, isomerization, and bond rearrangements of amide-containing amino acids and also occurs as a side reaction in peptide synthesis. The CTL specifically recognized the succinimide-containing peptide showing only weak cross-reactivity at high concentrations of the parent peptide containing unmodified asparagine. Similarly, CTL raised against the parent peptide did not recognize the succinimide derivative of this peptide. Naturally processed forms of these structures are likely to occur given the importance and frequency of deamidation both in vitro and in vivo. Moreover, since succinimide intermediates of deamidated peptides can occasionally be very stable, these peptides have the potential to act as altered self-Ags with significant implications for autoimmunity. In addition, unwanted and potentially hazardous specificities may be elicited when using synthetic peptides in subunit vaccines in which succinimide residues may form spontaneously during storage or chemical synthesis.
Activation of murine lymphocytes by lipopolysaccharide incorporated in fusogenic, reconstituted influenza virus envelopes (virosomes).
Authors: Dijkstra J , Bron R , Wilschut J , de Haan A , Ryan J L
Published: December 2022

We have studied the in vitro activation of murine lymphocytes with LPS incorporated in the membranes of both phospholipid vesicles (liposomes) and vesicles composed of fusogenic, reconstituted influenza virus envelopes (virosomes). The incorporation of Salmonella minnesota rough-LPS in liposomes reduced the potency of LPS to stimulate splenocyte proliferation and cell surface kappa-light chain expression on 70 Z/3 pre-B cells by over 100-fold. Salmonella minnesota rough-LPS inserted into virosomes was at least 10-fold more potent than free LPS, both when prebound virosomes were allowed to be taken up by the cells at neutral pH and when the virosomes were fused into the plasma membrane by low pH treatment. Inactivation of the virosomes by low pH pretreatment reduced the potency of the virosomal LPS to the level of liposome-incorporated LPS. The association of the various LPS forms with the cells was quantitated using radio-iodinated LPS. Correcting for uptake, virosomal LPS remained 2- to 10-fold more potent than free LPS in stimulating B lymphocytes and at least 100-fold more active than liposomal LPS or fusion-inactivated virosomes. After low pH-induced fusion with the plasma membrane, the majority (80%) of the prebound virosomes had fused with the cells, compared with about 8% after neutral uptake. From these results we conclude that LPS inserted into the plasma or endosomal membranes efficiently activates murine lymphocytes. The fusion data suggest that the incorporation into endosomal membranes might be a more effective stimulus.
bcl-2 alters the antigen-driven selection of B cells in mukappa but not in mu-only Xid transgenic mice.
Authors: Kenny J J , Fischer R T , Lustig A , Dintzis H , Katsumata M , Reed J C , Longo D L
Published: December 2022

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.
Cross-reactivity of T cell lines and clones to beta-lactam antibiotics.
Authors: Mauri-Hellweg D , Zanni M , Frei E , Bettens F , Brander C , Mauri D , Padovan E , Weltzien H U , Pichler W J
Published: December 2022

To clarify on a molecular level the specific T cell response to haptens like penicillin G, we generated T cell lines and clones from penicillin-allergic patients. Two types of beta-lactam reactivity of T cells could be delineated: one group of patients showed a rather restricted specificity, as the penicillin-elicited T cell lines generated from such donors proliferated only to the stimulating penicillin, but not to other beta-lactam antibiotics nor to cephalosporines, even if the side chain was identical. This indicates that the penicilloyl structure together with the side chain was recognized by these T cells. The second group comprised patients with more broadly reactive T cells, as they were restimulated by penicillin G as well as by related penicillins like amoxicillin or ampicillin, but not cephalosporines. This indicates that the penicilloyl structure, a common motif of penicillins, was important for T cell recognition. Clones generated from a broadly reactive patient confirmed this heterogeneity, as either monospecific or broadly specific T cell clones could be identified. This broad or very restricted pattern of T cell reactivity was reflected in the use of TCR Vbeta-chains: while the broadly reactive T cell lines showed a heterogenous TCR usage, the highly restricted T cell lines showed an up-regulation of one TCR Vbeta-chain. Thus, our data suggest that the outgrowth of T cells bearing a certain TCR Vbeta may be a sign of a limited cross-reactivity.
IL-12 induces human T cells secreting IL-10 with IFN-gamma.
Authors: Windhagen A , Anderson D E , Carrizosa A , Williams R E , Hafler D A
Published: December 2022

A clear differentiation of Th1 and Th2 cytokine-secreting subsets in humans has not yet been defined. To further examine cytokine-directed differentiation of human T cell responses to both exogenous and autoantigens, we generated 346 short term T cell lines at limiting dilutions from six normal individuals to tetanus toxoid and myelin basic protein in the presence of IL-2 with or without the addition of IL-12 and anti-IL-4 mAb. T cell lines were examined for [3H]thymidine incorporation and cytokine secretion of IFN-gamma, IL-4, and IL-10. After culture in the presence of IL-12 and anti-IL-4 mAb, the predominant T cell response to Ag stimulation was simultaneous secretion of IL-10 and IFN-gamma. The concomitant secretion of IL-10 and IFN-gamma by T cells was confirmed by stimulating lines in the absence of APCs with plate-bound anti-CD3 mAb after two rounds of Ag-specific stimulation. Moreover, IL-12 enhanced IL-10 and IFN-gamma production in a myelin basic protein-reactive T cell clone, demonstrating that a differentiated T cell clone could be induced to secrete both cytokines. The addition of a neutralizing anti-IFN-gamma Ab to cultures with IL-12 and anti-IL-4 mAb during the generation of tetanus toxoid-reactive lines had no effect on the induction of IL-10 and IFN-gamma secretion, indicating that IL-12 and not IFN-gamma was responsible for the induction of this subset of T cells. Thus, in human T cells, IL-12 induces concomitant secretion of IL-10 and IFN-gamma.
Chemokine expression by intraepithelial gamma delta T cells. Implications for the recruitment of inflammatory cells to damaged epithelia.
Authors: Boismenu R , Feng L , Xia Y Y , Chang J C , Havran W L
Published: December 2022

T cells expressing gamma delta TCR may have evolved to recognize Ag in a different manner as well as perform a broader set of functions than T cells with alpha beta TCR. In this study, we tested the hypothesis that dendritic epidermal T cells (DETC) bearing the invariant V gamma 3V delta 1 TCR may be able to signal the migration of peripheral alpha beta T cells to the epidermis by secreting specific chemokines. Expression of macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, RANTES, and lymphotactin was inducible in DETC 7-17 cells, whereas mRNA for monocyte chemoattractant protein (MCP)-1 could not be detected. Strikingly, lymphotactin was the most abundant chemokine produced by activated DETC 7-17 cells. Activated primary DETC cultures also produced copious amounts of lymphotactin mRNA. Similarly, freshly isolated and activated intestinal intraepithelial T cells (i-IEL) with gamma delta TCR expressed high levels of lymphotactin mRNA. In contrast, lymphotactin mRNA was present in activated spleen gamma delta T cells at low basal levels. Migration of CD8+ T cells induced by culture supernatants from stimulated DETC 7-17 cells was strongly reduced in the presence of a neutralizing anti-lymphotactin antiserum and to a lesser extent by neutralizing anti-MIP-1 alpha, anti-MIP-1 beta, or anti-RANTES antiserum. The presence of lymphotactin in supernatants from activated DETC 7-17 cultures was directly demonstrated by Western blot analysis. These observations are consistent with a model in which gamma delta IEL play an active multi-faceted role in the maintenance of epithelia homeostasis.
Implantation of IL-2-containing osmotic pump prolongs the survival of superantigen-reactive T cells expanded in mice injected with bacterial superantigen.
Authors: Kuroda K , Yagi J , Imanishi K , Yan X J , Li X Y , Fujimaki W , Kato H , Miyoshi-Akiyama T , Kumazawa Y , Abe H , Uchiyama T
Published: December 2022

In the present study we investigated the mechanism of deletion of superantigen (sAg)-reactive T cells expanded in sAg-injected mice. In staphylococcal enterotoxin A (SEA)-injected mice, IL-2 activity in serum peaked at 1 to 3 h and the expression of IL-2R alpha-chain (IL-2R alpha) on SEA-reactive (V beta 3+, or V beta 11+) T cells peaked at 6 to 12 h after the injection. Expansion of V beta 3+ or V beta 11+ T cells peaked at 2 days after the injection when most of these T cells were IL-2R alpha negative, and IL-2 activity was not detected at all in serum, suggesting the involvement of IL-2 deprivation in the deletion of expanded T cells. Implantation of an osmotic pump containing human rIL-2 (IL-2 pump) prolonged the expanded states of V beta 3+ or V beta 11+ T cells in SEA-injected C57BL/6 mice and of V beta 8+ T cells in SEB-injected MRL +/+ and Fas Ag-defective MRL-Ipr/Ipr mice. Adult thymectomy did not change at all the effect induced by implantation of IL-2 pump. DNA fragmentation was blocked substantially in mice co-treated with SEA and IL-2 pump. In addition, CD4+ T cell blasts, obtained by in vitro stimulation with rIL-2 of splenic CD4+ T cells from mice co-treated with SEA and IL-2 pump, produced substantial amounts of IL-2 upon restimulation with SEA. These results indicate that deprivation of IL-2 is deeply involved in the deletion of expanded sAg-reactive T cells and their anergy induction in sAg-injected mice.
Tyrphostin A9 inhibits calcium release-dependent phosphorylations and calcium entry via calcium release-activated channel in Jurkat T cells.
Authors: Marhaba R , Mary F , Pelassy C , Stanescu A T , Aussel C , Breittmayer J P
Published: December 2022

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.
Cytokine activation sensitizes human monocytic and endothelial cells to the cytotoxic effects of an anti-CD40 immunotoxin.
Authors: Francisco J A , Kiener P A , Moran-Davis P , Ledbetter J A , Siegall C B
Published: December 2022

G28-5 sFv-PE40 is a single-chain immunotoxin that is cytotoxic toward malignant B cells expressing CD40. Human monocytes, which also express cell surface CD40, were found to be insensitive to the immunotoxin. Activation of the monocytic cell line THP-1, or freshly isolated peripheral blood monocytes with IFN-gamma, but not IL-3, GM-CSF, IL-6, or TNF-alpha, greatly sensitized the cells toward G28-5 sFv-PE40, lowering the EC50 value from > 10 micrograms/ml to 80 ng/ml. This sensitization could not be explained simply by the two- to threefold increase in cell surface CD40 expression induced by IFN-gamma since TNF-alpha or the combination of TNF-alpha and IL-6 gave similar increases in CD40 expression but did not sensitize the cells to the immunotoxin. Internalization of G28-5 sFv-PE40 after IFN-gamma activation was also increased threefold, reflective of the increase in CD40 expression. IFN-gamma-treated but not -untreated THP-1 cells produced IL-6 and TNF-alpha following incubation with G28-5 sFv-PE40, indicating an association between CD40 signaling, which induces cytokine production, and sensitivity to the immunotoxin. HUVECs also express CD40 but were found to be insensitive to the anti-CD40 immunotoxin. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, sensitized the endothelial cells to G28-5 sFv-PE40. These data show that activation with cytokines can sensitize monocytes and endothelial cells to an immunotoxin targeted to CD40, most likely by altering the trafficking and/or processing of the immunotoxin after receptor-mediated internalization.
The central nervous system as an immunocompetent organ: role of glial cells in antigen presentation.
Authors: Shrikant P , Benveniste E N
Published: December 2022

The central nervous system has the capacity to initiate and regulate immune responses by allowing restricted entry of peripheral immune cells and providing a microenvironment that is conducive to the activation of immune effector cells. In this commentary, we discuss the ability of two glial cell types, astrocytes and microglia, to function as APCs. In particular, we focus on the phenotypic and functional distinctions between astrocytes and microglia that may contribute to their differing abilities to effectively present Ag to CD4+ cells.