bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of adjuvant arthritis in Lewis rats by oral administration of type II collagen.
Authors: Zhang Z Y , Lee C S , Lider O , Weiner H L
Published: December 2022

Adjuvant arthritis is induced by intradermal injection of Mycobacterium tuberculosis (MT) in oil. The role of immunity to type II collagen (CII) in adjuvant arthritis (AA) has not been well defined. We found that oral administration of chicken CII given 3 micrograms per feeding on days -7, -5, and -2 before disease induction consistently suppressed the development of AA. A decrease in delayed-type hypersensitivity responses to CII was also observed that correlated with suppression of AA. AA was optimally suppressed by 3 and 30 micrograms of collagen type II variably by 300 micrograms, and not by 0.3 microgram or 1 mg. Oral administration of collagen type I also suppressed AA; only minimal effects were seen with collagen type III. Suppression was Ag specific: feeding CII did not suppress experimental autoimmune encephalomyelitis; feeding myelin basic protein suppressed experimental autoimmune encephalomyelitis, but not AA. Suppression of AA could not be consistently obtained by feeding MT. Suppression of AA could be adoptively transferred by T cells from CII fed animals and could be obtained when CII was fed after disease onset. Our results suggest that autoimmunity to CII has a pathogenic role in AA and raise the possibility that cross-reactive epitopes exist between CII and MT. Alternatively, the pathogenesis of AA may be dependent on developing immunity to CII. These results further demonstrate the effectiveness of oral tolerance as a means to suppress experimental autoimmune diseases.
Self-binding antibodies (autobodies) form specific complexes in solution.
Authors: Kaveri S V , Halpern R , Kang C Y , Köhler H
Published: December 2022

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.
Analysis of the primary signals required for activation of the mitogenic pathway in murine thymocytes from protein phosphorylation patterns.
Authors: Hesketh R , Moore T A , Pennington S R , Smith G A , Metcalfe J C
Published: December 2022

It has been proposed that phorbol esters and the Ca2+ ionophore A23187 are effective comitogens for some species of lymphocytes because together they mimic the normal secondary signals for cell activation by mitogens that cause phosphatidylinositol 4,5-bisphosphate (PtdInsP2) breakdown (e.g., anti-TCR and anti-Thy-1 antibodies and Con A). To test whether activation of protein kinase C and an increase in [Ca2+]i account for the activation of the mitogenic pathway in murine thymocytes by the mitogens that cause PtdInsP2 breakdown, the two-dimensional phosphorylation patterns generated by the three classes of mitogens (protein kinase C activator, Ca2+ ionophore, and activator of PtdInsP2 breakdown) and by activators of cAMP-dependent kinases have been compared. From the phosphorylation patterns, by which each mitogen could be distinguished reproducibly, it was concluded that: 1) The phosphorylation patterns generated by the mitogens that activate PtdInsP2 breakdown are only slightly affected by the removal of extracellular Ca2+ under conditions that abolish the normal rise in [Ca2+]i and do not therefore depend on the activation of Ca2(+)-dependent protein kinases. In contrast, the phosphorylation pattern generated by A23187 is totally dependent on extracellular Ca2+. 2) Neither A23187 nor the mitogens that activate PtdInsP2 breakdown nor activators of cAMP-dependent kinases caused significant activation of protein kinase C assayed by phosphorylation of the diagnostic proteins 80b and 78a. Consistent with this conclusion, only the phorbol esters or oleoyl acyl glycerol caused translocation of protein kinase C activity from the cytosolic to the membrane fraction. 3) Neither A23187 nor the mitogens that cause PtdInsP2 breakdown activated cAMP-dependent kinases. Taken together the data imply that the mitogens that cause PtdInsP2 breakdown must generate an additional, independent primary mitogenic signal. It is suggested that this signal may be the activation of tyrosine kinases (e.g., p56lck) via the TCR and working hypotheses for effective combinations of primary mitogenic signals that will activate DNA synthesis are developed.
Platelet-activating factor enhances Ig production in B lymphoblastoid cell lines.
Authors: Mazer B , Clay K L , Renz H , Gelfand E W
Published: December 2022

Platelet-activating factor (PAF) is a highly potent phospholipid mediator known to be active in many biologic systems. To date, little is known of the effect of PAF on B lymphocytes. Using two Ig-secreting B lymphoblastoid cell lines, we have demonstrated that PAF can enhance Ig production by these cells in a dose-dependent fashion. PAF also causes significant alteration of the kinetics of Ig secretion in these lymphoblastoid cell lines. The effect of PAF is rapid, with detection of 6- to 12-fold increases in Ig production in the first 24 h of cell culture, followed by a plateau during the next 24 to 48 h. The specificity of the PAF effect on Ig secretion is emphasized by lyso-PAF having no Ig-enhancing properties and by the inhibition of Ig enhancement in the presence of the structural analogue PAF antagonist CV3988 and the soluble nonstructural analogue PAF receptor antagonist Web 2086. PAF does not cause an increase in the kinetics of cell proliferation or an increase in cell numbers at any time during a 72-h culture period. In an attempt to explain the increase in Ig secretion in the absence of changes in growth parameters, an ELISA spot assay for enumeration of Ig-secreting cells was developed. This assay demonstrated that the increase in Ig production is likely due to enhancement of single cell Ig secretion rather than an increase in cell number. These data indicate that PAF may have an important immunomodulatory role in the production of Ig by B lymphocytes.
Two pathways of CD11b/CD18-mediated neutrophil aggregation with different involvement of protein kinase C-dependent phosphorylation.
Authors: Merrill J T , Slade S G , Weissmann G , Winchester R , Buyon J P
Published: December 2022

The characteristics of homotypic neutrophil aggregation, mediated by the adhesion molecule CD11b/CD18, differ according to whether activation takes place via intracellular protein kinase C(PKC) inducers or chemoattractants. In response to diacylglycerol (DAG) analogues such as PMA and 1,2-dioctanoyl-sn-glycerol, a prolonged cellular aggregation occurs that is associated with intense phosphorylation of the CD18 beta-chain. In response to the chemoattractant FMLP, a more transient aggregation event results that is associated with minimal beta-chain phosphorylation. By using the PKC inhibitor staurosporine, we now show that these differences are likely to reflect two different pathways of activation. Both aggregation and phosphorylation induced by DAG analogues are completely abolished by staurosporine in a parallel dose-dependent manner. Conversely, FMLP-induced aggregation is enhanced and prolonged by staurosporine whereas the associated minimal phosphorylation event is further diminished by staurosporine. Accordingly, activation of neutrophil aggregation by DAG analogues is associated with and presumably due to phosphorylation of the CD18 beta-chain. This intense phosphorylation occurs via a staurosporine-sensitive kinase such as PKC. FMLP, on the other hand, appears to activate CD11b/CD18 by a distinct mechanism. This latter mechanism does not seem to be dependent on what may be a minor PKC-induced phosphorylation of the beta-chain, and indeed is enhanced by inhibition of PKC. Of note, staurosporine was also found to cause selective release of specific granules with concomitant increase in surface display of CD11b/CD18. These data further support previous observations that up-regulation of this adhesive molecule is not the primary event in the induction of cellular adhesiveness.
Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo.
Authors: Nourshargh S , Williams T J
Published: December 2022

The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.
Regulation of HIV replication in infected monocytes by IFN-alpha. Mechanisms for viral restriction.
Authors: Gendelman H E , Baca L M , Turpin J , Kalter D C , Hansen B , Orenstein J M , Dieffenbach C W , Friedman R M , Meltzer M S
Published: December 2022

In a survey of 15 different virus isolates, no IFN-alpha or IFN-beta activity was detected in culture fluids of HIV-infected T cells or monocytes. Exogenous rIFN-alpha added to T lymphoblast or monocyte cultures induced restriction in replication of the amphotropic HIV that infect both cell types. With IFN-treated HIV-infected T cells, levels of reverse transcriptase (RT) activity in culture fluids were half those in control cultures, but the frequency of infected cells or the levels of p24 Ag released in culture fluids were unchanged. In contrast to the modest effect of IFN on HIV-infected T cells, IFN-induced antiviral activity in monocytes was quite dramatic. Monocytes treated with IFN at the time of virus challenge showed no evidence of HIV infection: no p24 Ag or RT activity, no viral mRNA, and no proviral DNA. In this system, IFN interrupts one or more early event(s) in the virus replication cycle before formation of proviral DNA. Monocyte cultures infected with HIV 7 days before IFN treatment showed a gradual decrease in levels of p24 Ag and RT activity to baseline by 3 wk. HIV-induced cytopathic changes were markedly reduced, and the frequency of productively infected cells was less than or equal to 1% of total cells. Virus particles released 24 h after IFN treatment were 100- to 1000-fold less infectious than equal numbers of control virions. But, monocytes treated with IFN 7 days after HIV infection were not free of the retroviral pathogen: levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.
Synthetic peptides containing T and B cell epitopes from human immunodeficiency virus envelope gp120 induce anti-HIV proliferative responses and high titers of neutralizing antibodies in rhesus monkeys.
Authors: Hart M K , Palker T J , Matthews T J , Langlois A J , Lerche N W , Martin M E , Scearce R M , McDanal C , Bolognesi D P , Haynes B F
Published: December 2022

We have previously described a synthetic peptide (T1-SP10) derived from two noncontiguous regions of HTLVIIIB envelope gp120 (T1, amino acids 428-443; SP10, amino acids 303-321) that induced type-specific anti-HIV neutralizing antibodies and T cell proliferative responses against native HIV gp120 when used as a carrier-free immunogen in goats. In this study, HTLVIIIB T1-SP10 synthetic peptides were used to immunize rhesus monkeys to determine if the peptides were capable of eliciting HIV-specific neutralizing antibody and proliferative responses in primates. Four compounds (alum, polyA:polyU, threonyl-muramyldipeptide (MDP) and IFA) were also compared for efficacy as adjuvants in this system. Rhesus monkeys immunized with T1-SP10 peptides generated high titers of antibodies against the immunogens and also against HTLVIIIB gp120. Sera from all four animals given T1-SP10 in IFA or threonyl-MDP neutralized infection by HTLVIIIB and blocked virus-dependent cell fusion events. A peak neutralization titer of 1:940 was seen in one animal given IFA (19600) and a titer of 1:900 was seen in one of the monkeys (17371) given threonyl-MDP. Proliferative responses of immune rhesus PBMC to T1-SP10 appeared after the first injection. After eight immunizations, two of eight monkeys (one injected with peptides in threonyl-MDP and one given peptides in IFA) had PBMC proliferative responses to native HTLVIIIB gp120. These data demonstrate that the carrier-free T1-SP10 synthetic peptide construct can induce high titers of neutralizing anti-HIV antibody responses and PBMC proliferative responses to HIV in primates.
Contribution of the CD5+ B cell to D-proximal VH family expression early in ontogeny.
Authors: Jeong H D , Teale J M
Published: December 2022

In this study, the contribution of the CD5+ B cell to the preferential expression of VH 7183 and Q52 observed early in development was determined. CD5+ and CD5- B cells from BALB/c mice were isolated by fluorescence-activated cell sorter and the expression of particular VH gene families was determined directly by in situ hybridization. The results indicate that CD5+ B cells obtained from both adult and neonatal animals express Q52 at increased levels compared with CD5- B cells. Preferential expression of VH 7183 was observed only in the neonatal CD5- B cell subset. Thus, the increased expression of VH 7183 early in development is caused by the CD5- subset whereas increased Q52 expression is caused by the CD5+ subset. These results indicate that the fetal/neonatal conventional B cell is distinct from conventional adult B cells in terms of Ig gene repertoire expression.
Rapid nonlysosomal degradation of assembled HLA class II glycoproteins incorporating a mutant DR alpha-chain.
Authors: Koppelman B , Cresswell P
Published: December 2022

Gamma irradiation followed by antibody and complement selection was used to isolate a human B-lymphoblastoid cell line that no longer expresses HLA-DR molecules on its cell surface. Cell surface expression in the mutant (HMy2.DRN) was restored by transfecting a wildtype DRA but not a DRB cDNA, suggesting that a structural mutation in the DRA mRNA or protein was responsible for the lack of cell surface expression. Nucleotide sequence analysis of the DRA mRNA from HMy2.DRN revealed a 75 nucleotide deletion corresponding to the start of the alpha 2 domain and involving one of two cysteines that are involved in the formation of an intrachain disulfide bond. At the biochemical level, only minute quantities of HLA-DR could be precipitated from this cell line after a 4-h continuous label with 35S-methionine. HLA-DR beta and the class II-associated invariant chain could be seen coprecipitating with the mutant DR alpha-chain, suggesting a limited accumulation of normally assembled molecules. However, by carrying out the labeling at 16 degrees C instead of 37 degrees C, equivalent amounts of HLA-DR could be precipitated from parent and mutant alike. The mutant DR alpha chain was found in association with the beta-chain, but with reduced association with the invariant chain under these conditions. Pulse chase analysis in the parent and mutant cell lines indicated that this mutant DR alpha beta I complex undergoes a process of degradation at 37 degrees C. Inhibitors of intracellular transport such as monensin were ineffective in blocking this process of degradation. This work is consistent with published reports implicating the involvement of a pre-Golgi or an early Golgi compartment in the proteolysis of aberrantly folded or assembled multisubunit proteins.
Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells.
Authors: Chang T L , Shea C M , Urioste S , Thompson R C , Boom W H , Abbas A K
Published: December 2022

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.
Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte.
Authors: Marucha P T , Zeff R A , Kreutzer D L
Published: December 2022

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.
The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM.
Authors: Klein D P , Galea S , Jongstra J
Published: December 2022

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.
Analysis of the immune response to lipopolysaccharide. Existence of an interspecies cross-reactive idiotype associated with anti-lipid A antibodies.
Authors: Su S D , Ward M M , Apicella M A , Ward R E
Published: December 2022

LPS is the major surface glycolipid on gram-negative bacteria. In this work, we have idiotypically characterized the antibody response against LPS in different species. To do this, we have produced mAb against LPS. Binding of many of these antibodies to LPS could be inhibited by LPS and lipid A, indicating that the monoclonals are specific for lipid A, the toxic moiety of the LPS molecule. One anti-lipid A antibody, IC9, proved protective against gram-negative bacteremia and endotoxic shock in murine protection models. We generated anti-idiotypic antibodies against IC9. The binding of several of these anti-Id to IC9 was specifically inhibited by lipid A. We used these anti-Id to characterize the anti-LPS response, and the results revealed that the IC9 Id is conserved in different species. The importance of an interspecies cross-reactive Id in the response to endotoxin and its relevance in vaccine development for septic shock are discussed.
Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils.
Authors: Hébert C A , Luscinskas F W , Kiely J M , Luis E A , Darbonne W C , Bennett G L , Liu C C , Obin M S , Gimbrone M A , Baker J B
Published: December 2022

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.
An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.
Authors: Fleischer B , Gerardy-Schahn R , Metzroth B , Carrel S , Gerlach D , Köhler W
Published: December 2022

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.
Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.
Authors: Landis R C , Friedman M L , Fisher R I , Ellis T M
Published: December 2022

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.
Macrophage activation for intracellular killing as induced by calcium ionophore. Correlation with biologic and biochemical events.
Authors: Buchmüller-Rouiller Y , Mauël J
Published: December 2022

Changes in the concentration of cytosolic Ca2+ are known to affect various macrophage functions; in particular, exposure in vitro to the Ca2+ ionophore A23187 primes macrophages for tumor cell killing. In the present report, it is shown that treatment with this ionophore similarly mimics IFN-gamma as a priming signal for induction of microbicidal activity. Incubation of mouse bone marrow-derived macrophages with 10(-7) to 10(-6) M A23187 (in the presence of Ca2+) led to intracellular killing of the protozoan parasite Leishmania enriettii within 24 h, provided LPS (1 ng/ml) was also present; no microbicidal activity was observed using either compound alone. A 4-h exposure to the ionophore in the presence of Ca2+ (priming phase) was sufficient to induce leishmanicidal activity upon reincubation with LPS, here acting as a necessary second signal. Addition of EGTA during the priming phase blocked intracellular killing upon subsequent LPS treatment; microbicidal activity could be restored by excess Ca2+, but not Mg2+, suggesting that changes in the concentration of cytosolic Ca2+ are sufficient to mediate the molecular events that lead to acquisition of microbicidal potential. Ionophore-induced leishmanicidal activity was paralleled by a stimulation of the hexosemonophosphate shunt pathway and production of nitrites, which are biochemical correlates of the activated state. In addition, sequential exposure to A23187 and LPS markedly stimulated macrophages to release TNF and PGE2, two agents thought to act as modulators of macrophage activation.
Moderate zinc deficiency in rhesus monkeys. An intrinsic defect of neutrophil chemotaxis corrected by zinc repletion.
Authors: Vruwink K G , Fletcher M P , Keen C L , Golub M S , Hendrickx A G , Gershwin M E
Published: December 2022

Experimental animals fed zinc-deficient diets are well known for susceptibility to infections and impaired mitogen response and Ig production. However, the levels of zinc deficiency used have generally been severe, not comparable to human populations, and have not addressed neutrophil function. To address this issue we have studied the effect in rhesus monkeys of a well defined moderately zinc-deficient (MZ) diet on polymorphonuclear leukocyte (PMN) function. Female adult rhesus monkeys were fed either a control (100 micrograms Zn/g) or MZ (2 micrograms Zn/g) diet for 9 mo with quantitation of PMN chemotaxis, and phagocytosis of opsonized yeast. In addition, membrane potential and secretion responses (changes in 90 degrees light scatter) and changes in PMN shape (forward light scatter shifts) were also measured. When compared to the PMN of animals fed control diets, there was a significant reduction in chemotaxis to FMLP of MZ-fed monkey PMN. Although shape change, cell membrane depolarization, as well as phagocytosis were not significantly different among the two groups, the PMN of MZ animals had significantly lower relative loss of orthogonal light scatter (degranulation) due primarily to a lower resting orthogonal light scatter and also a smaller loss when stimulated with FMLP. In vitro addition of zinc to the cells (25 microM) did not improve chemotaxis, and in fact, was inhibitory for most control and zinc-deficient cells. However, after 2 wk of dietary zinc repletion (100 micrograms Zn/g), chemotaxis in the low zinc group was higher and comparable to the control response. These data indicate that zinc deficiency is associated with an intrinsic PMN defect that specifically affects chemotaxis and is corrected with dietary zinc repletion.
Equivalent recognition of a varicella-zoster virus immediate early protein (IE62) and glycoprotein I by cytotoxic T lymphocytes of either CD4+ or CD8+ phenotype.
Authors: Arvin A M , Sharp M , Smith S , Koropchak C M , Diaz P S , Kinchington P , Ruyechan W , Hay J
Published: December 2022

Immunity to varicella-zoster virus (VZV), a member of the alpha-herpes virus family, exemplifies the host response to an ubiquitous human viral pathogen. In this investigation of the cytotoxic T lymphocyte (CTL) response to VZV, the depletion of CD4+ T lymphocytes made it possible to demonstrate CD8(+)-mediated cytotoxic function against autologous VZV-infected lymphoblastoid cells targets. CTL recognition of two major VZV proteins, the immediate early protein (IE62) and gp I, was demonstrated in limiting dilution cultures of T lymphocytes obtained from immune donors, stimulated with inactivated VZV Ag, and tested against lymphoblastoid cells infected with vaccinia recombinants expressing these VZV proteins. Among 11 VZV donors tested at least 20 y after primary infection, the mean precursor frequency for T lymphocytes that recognized the IE62 protein was 1:105,000 +/- 85,000 SD, with a range of 1:13,000 to 1:231,000. The mean frequency of CTL precursors specific for gp I in 11 subjects was equivalent, with a mean of 1:121,000 +/- 86,000 SD (range 1:15,000 to 1:228,000) (p = 0.68). Limiting dilution cultures were also prepared using purified CD4+ or CD8+ T lymphocyte populations recovered from PBMC by sterile fluorescence-activated cell sorting. CTL precursors that recognized the IE62 protein or gp I were derived from each of the major T lymphocyte populations by stimulation with inactivated VZV Ag; CD4+ and CD8+ CTL precursor frequencies for the IE62 protein and gp I were equivalent (p = 0.2). We conclude that antiviral CTL activity against targets expressing VZV proteins was mediated equally well by T lymphocytes of the CD4+ or CD8+ phenotype and that antiviral CTL function could be elicited in each subpopulation by exposure to non-infectious viral Ag.
Phagocytosis of Leishmania enhances macrophage activation by IFN-gamma and lipopolysaccharide.
Authors: Corradin S B , Mauël J
Published: December 2022

This investigation describes the ability of Leishmania promastigotes to enhance activation of bone marrow-derived murine macrophages in vitro if added together with rIFN-gamma in the presence or absence of LPS. Activation was defined as the capacity for arginine-derived NO2- production and the killing of intracellular Leishmania. Enhanced NO2- production was observed for either CBA or C3H/HeJ macrophages undergoing phagocytosis at the time of activation. Other phagocytic stimuli including inert polystyrene latex beads were as effective as Leishmania. No correlation could be demonstrated between the enhanced NO2- release and secretion of products of the respiratory burst or PGE2. However, TNF-alpha secretion was elevated in cultures undergoing phagocytosis and a relationship between hexosemonophosphate shunt activity and NO2- levels was evident. These studies confirm and extend previous reports that phagocytosis plays an important role in the regulation of macrophage physiology.
The role of cytophilic IgG3 antibody in T cell-mediated resistance to infection with the extracellular bacterium, Pseudomonas aeruginosa.
Authors: Markham R B , Pier G B , Schreiber J R
Published: December 2022

Previous studies have demonstrated that T lymphocytes from mice immunized with a high m.w. polysaccharide Ag from Fisher-Devlin immunotype I Pseudomonas aeruginosa can adoptively transfer protection against challenge with the homologous bacterial strain to susceptible mice. This T cell-mediated resistance has been found to be B cell dependent, although serum from immunized mice is incapable of passively transferring protection to nonimmune mice. The current studies demonstrate that T cells from immunized mice possess receptors that permit them to be adsorbed to IgG3-secreting hybridomas, but not to IgM-secreting hybridomas. Cross-linking of antibody on the surface of immune T cells results in release of a soluble factor that inhibits bacterial growth. Treatment of T cells to remove cytophilic antibody eliminates their ability to adoptively transfer protection to nonimmune mice, and the protective ability can be restored by co-incubating the T cells with monoclonal P. aeruginosa-specific IgG3 antibody before adoptive transfer to nonimmune mice. These observations are consistent with a model in which T lymphocytes from immunized mice are activated by cross-linking of FcR for IgG3 to secrete an antibacterial lymphokine. The ability of IgG3 at low antibody concentrations to act synergistically with T lymphocytes to inhibit bacterial growth could explain the evolutionary selection of this antibody isotype as the predominant subclass of IgG secreted in response to bacterial capsular polysaccharide Ag.
T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.
Authors: Janssen O , Wesselborg S , Heckl-Ostreicher B , Pechhold K , Bender A , Schondelmaier S , Moldenhauer G , Kabelitz D
Published: December 2022

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.
Monoclonal antibody against the human peripheral lymph node homing receptor homologue (Leu 8) inhibits B cell differentiation but not B cell proliferation.
Authors: Murakawa Y , Strober W , James S P
Published: December 2022

Previous studies have shown that a subpopulation of circulating human B cells expresses the Leu 8 peripheral lymph node homing receptor homologue and that these B cells are capable of producing Ig in response to staphylococcus A Cowan I (SAC). In the present study the effect of a signal delivered via the Leu 8 molecule (using anti-Leu 8 mAb) on B cells was examined. Initially, it was shown that immobilized anti-Leu 8 suppressed IgM and IgG secretion of B cells activated by SAC + IL-2 but not that by PWM-prestimulated B cells or B cells stimulated with PWM in the presence of CD4+, Leu 8- T cells (a source of helper cells). It was also shown that anti-Leu 8 did not suppress SAC + IL-2-stimulated B cell proliferation or expression of IL-2R alpha-chain or c-myc mRNA in B cells. The addition of T cells, monocytes, purified IL-2, rIL-1, rIL-6, or human B cell growth factor did not overcome the inhibitory effect of anti-Leu 8 on SAC-stimulated B cell Ig production, and the inhibitory effect of anti-Leu 8 was not blocked by anti-TGF-beta. Finally, inhibition of B cell differentiation occurred even when anti-Leu 8 was added up to 72 hrs after initiation of cell culture. Thus, anti-Leu 8 is unique among inhibitors of B cell function in that it can down-regulate immunoglobulin synthesis without affecting B cell proliferation. These findings suggest that a natural ligand for Leu 8 could affect not only homing of B cells, but also B cell differentiation.
CD8+ T cell homing to the pancreas in the nonobese diabetic mouse is CD4+ T cell-dependent.
Authors: Thivolet C , Bendelac A , Bedossa P , Bach J F , Carnaud C
Published: December 2022

The adoptive transfer of type I diabetes in nonobese diabetic mice requires the contribution of both CD4+ and CD8+ T cells. To further elucidate the cellular pathway(s) of beta-cell destruction and the responsibility of each subset, high doses of committed T cells from diabetic mice purified to single subsets, were injected into syngeneic nonobese diabetic neonates. The recipients of single or mixed subsets were followed for clinical manifestations of diabetes and examined at 30 days of age for in situ lesions. None of the animals injected with either CD4+ or CD8+ T cells became overtly diabetic during the 30 days of observation whereas 8 of 23 mice inoculated with a mixture of the two subsets developed glycosuria and hyperglycemia. However, insulitis was found in 6 of the 13 mice injected with CD4+ T cells whereas only 1 of the 9 mice injected with CD8+ T cells showed marginal infiltration of the pancreas. The lesions initiated by CD4+ T cells alone were considerably less severe than those induced by the mixture of both subsets, corroborating the fact that overt disease did not occur in the former group. Together, these results suggest a distinct function for each diabetogenic T cell subset. CD4+ T cells, which have the capacity to home to the pancreas, promote in turn the influx of CD8+ effector T cells that do not by themselves accumulate in this organ. These results illustrate a novel form of T-T cell interactions leading to organ specific autoimmune lesions.
Anti-P autoantibody production requires P1/P2 as immunogens but is not driven by exogenous self-antigen in MRL mice.
Authors: Hines J J , Weissbach H , Brot N , Elkon K
Published: December 2022

Considerable evidence supports the idea that autoantibody production in human and murine SLE is Ag driven. To determine whether Ag (the ribosomal P proteins) could initiate autoantibody production in lupus mice, 34 MRL/lpr mice were immunized with mouse riboosomal proteins in Freund's adjuvant. Neither intact ribosomes, denatured total mouse ribosomal proteins, nor the purified mouse ribosomal proteins, P1 and P2, induced the production of anti-P autoantibodies in the MRL/lpr mice. In contrast to these negative findings, MRL/lpr mice immunized with Artemia salina ribosomes produced anti-P antibodies as well as anti-P autoantibodies. Although the induced anti-P autoantibodies bound exclusively to the carboxyl terminus, these anti-P antibodies differed from spontaneously occurring anti-P autoantibodies in their predominant binding to mouse P0 on immunoblots and their preferential reactivity against A. salina synthetic peptides by ELISA. Induction of anti-P antibodies required the presence of P1 and P2 on the ribosome because ribosomal cores devoid of P1 and P2 dimers did not induce anti-P. Despite the presence of approximately 80 ribosomal proteins, autoantibodies to other mouse ribosomal proteins were rarely observed. Immunization of MRL/+ mice and a normal H-2-matched strain of mice, C3H, also resulted in anti-P antibodies reactive with the A. salina P proteins and mouse P0. Whereas anti-P levels gradually declined in C3H mice, anti-P levels either remained elevated (MRL/lpr) or showed a secondary rise (MRL/+) at the onset of autoimmunity. These observations indicate that: i) high levels of autologous Ag are not sufficient to drive antiribosomal autoantibody production in MRL mice, ii) multivalency of the P proteins may explain their potent immunogenicity and ability to break tolerance, and iii) immunized MRL mice show an abnormal persistence of high level anti-P production presumably reflecting T cell activation of presensitized B cells.