bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS-induced sTNF-receptor release in vivo in a murine model. Investigation of the role of tumor necrosis factor, IL-1, leukemia inhibiting factor, and IFN-gamma.
Authors: Bemelmans M H , Gouma D J , Buurman W A
Published: December 2022

TNF, a primary mediator of the response to infection, can be injurious to the organism when present in excessive quantities. Circulating soluble TNF receptors (sTNFR) appear to represent a natural mechanism that protects against circulating TNF. Two soluble TNF receptors (sTNFR-P55 and sTNFR-P75) circulate in vivo and are up-regulated in response to endotoxin. In this study, we investigated the kinetics of LPS-induced sTNFR release and the role of the cytokines TNF, leukemia inhibiting factor, IFN-gamma, and IL-1 in this process. The results show that LPS injection results in a rapid increase in levels of both sTNFR. Although sTNFR-P55 decreases after a peak at 30 min, sTNFR-P75 levels show a peak after 4 to 8 h, after which they slowly diminish. Both human TNF and murine TNF are capable of increasing levels of both sTNFR. Blocking circulating TNF by administration of 3 different anti-TNF agents before LPS injection (mAb to murine TNF, sTNFR55-Fc or sTNFR75-Fc) results in a significant increase of sTNFR-P55 levels, whereas only both sTNFR-Fc constructs also significantly increase sTNFR-P75 levels. Although IL-1 receptor antagonist pretreatment before LPS has no effect on TNF or sTNFR levels, leukemia inhibiting factor pretreatment significantly increases sTNFR-P55 levels. Pretreatment with anti IFN-gamma mAb before LPS results in a significant reduction in TNF and sTNFR-P55 levels, but sTNFR-P75 levels are significantly increased. Our data show that both sTNFR can be up-regulated by LPS and TNF. The influence of TNF, leukemia inhibiting factor, IL-1, and IFN-gamma on the kinetics of LPS-induced circulating sTNFR is discussed in the context of the pathophysiology of LPS-induced disease.
In vivo characterization of the anti-inflammatory effect of cyclosporin A on human basophils.
Authors: Casolaro V , Spadaro G , Patella V , Marone G
Published: December 2022

We investigated whether cyclosporin A (CsA) in vivo exerts anti-inflammatory effects by inhibiting IgE- and non-IgE-dependent mediator release from human basophils. Six healthy volunteers were given oral CsA (5 mg/kg twice daily) or placebo for 5 days. Plasma CsA and basophil releasability in response to anti-IgE, FMLP, and A23187 were monitored 1 day before treatment, on alternate days during the treatment course, and 1 and 8 days after cessation of treatment. A constant plasma level of approximately 250 ng/ml CsA was obtained during CsA treatment. Basophil releasability in response to anti-IgE, FMLP, and A23187 was diminished by 20 to 60% throughout the course of CsA treatment. Placebo had no effect on basophil releasability. There was a significant correlation between plasma CsA and the decrease of histamine release induced by anti-IgE (rs = -0.66; p < 0.0005), FMLP (rs = -0.59; p < 0.001) and A23187 (rs = -0.68; p < 0.0001). In a second study, eight normal volunteers were given a single oral dose of CsA (7 mg/kg) or placebo, and plasma CsA and basophil releasability were monitored at different times thereafter. A rapid and significant reduction of histamine release induced by anti-IgE, FMLP, and A23187 paralleled a sharp increase of CsA plasma levels, which peaked at 5 h and lasted approximately 13 h. This study indicates that oral administration of CsA in normal subjects causes a rapid and significant inhibition of histamine release from basophils. This is the first evidence that in vivo administration of CsA can modulate the release of proinflammatory mediators from basophils obtained ex vivo.
Taxol shares the ability of bacterial lipopolysaccharide to induce tyrosine phosphorylation of microtubule-associated protein kinase.
Authors: Ding A , Sanchez E , Nathan C F
Published: December 2022

Microtubule-associated proteins may mediate the activation of macrophages by bacterial LPS. Three lines of evidence support this hypothesis: a) Taxol, a microtubule-binding diterpene, mimics the ability of LPS to induce cytokines and down-regulate receptors for TNF-alpha. In recombinant inbred mouse strains differing at the Lps gene, responsiveness to these effects of Taxol co-segregates with responsiveness to LPS. b) In vitro, LPS binds specifically to MT and preferentially to beta-tubulin. c) LPS activates microtubule-associated protein-2 kinase (MAPK). The present studies bring together and extend these lines of evidence. a) Taxol, like LPS, rapidly induces tyrosine phosphorylation of MAPK in mouse macrophages, and triggers MAPK to phosphorylate an exogenous substrate. b) Tyrosine phosphorylation of MAPK is an extremely rapid cellular response both to taxol and LPS. c) Macrophages from C3H/HeJ mice, which carry a defective Lps gene, fail to activate MAPK in response to taxol or LPS, although they activate MAPK in response to insulin or IFN-gamma. These results suggest that tyrosine phosphorylation of MAPK is among the earliest known response of macrophages to LPS. Taxol mimics LPS with respect to immediate MAPK activation, later transcriptional events, and the genetic control of both sets of responses. LPS and taxol thus appear to share an early step in a functionally important signal transduction pathway that may involve MT.
Tissue-specific regulation of IL-6 production by IL-4. Differential effects of IL-4 on nuclear factor-kappa B activity in monocytes and fibroblasts.
Authors: Donnelly R P , Crofford L J , Freeman S L , Buras J , Remmers E , Wilder R L , Fenton M J
Published: December 2022

IL-4 inhibits production of certain proinflammatory cytokines, including IL-1 beta, TNF-alpha, and IL-6, by activated monocytes. Although monocytes are a major source of IL-6, other cell types such as fibroblasts and endothelial cells can also express this cytokine. To determine whether IL-4 inhibits IL-6 expression in non-hemopoietic cells, we investigated the effects of IL-4 on IL-6 production in both primary human fibroblasts and fibroblast lines. Rheumatoid synovial fibroblasts were evaluated in these studies because, like monocytes, they produce high levels of IL-6 when stimulated with IL-1. Although peripheral blood monocytes did not constitutively express IL-6 mRNA or protein, stimulation with IL-1 or LPS induced de novo IL-6 expression in these cells. In contrast, synovial fibroblasts displayed a significant basal level of IL-6 production, which was markedly increased after stimulation with IL-1. IL-4 suppressed IL-6 expression in monocytes, but did not inhibit IL-6 production in synovial fibroblasts. The inability of IL-4 to suppress IL-6 synthesis in rheumatoid synovial fibroblasts was not caused by a lack of IL-4R and was not unique to these cells because IL-4 also failed to inhibit IL-6 production in normal fibroblast lines derived from other tissues. Inhibition of IL-6 production by IL-4 in monocytes was associated with decreased nuclear NF-kappa B levels. However, IL-4 does not globally suppress the activity of all DNA-binding proteins because IL-4 treatment did not reduce the levels of NF-IL-6 or NF-IL-1 beta B in the same cells. Because NF-kappa B activation is required for transcription of many cytokine genes, including IL-6, the ability of IL-4 to suppress NF-kappa B activity in monocytes suggests a potential mechanism by which this molecule may inhibit the expression of multiple cytokines.
Regulation of CD11b/CD18 expression in human neutrophils by phospholipase A2.
Authors: Jacobson P B , Schrier D J
Published: December 2022

Recent evidence suggests that phospholipase A2 (PLA2)-derived lipid mediators may regulate a number of neutrophil responses including degranulation and adhesion. In view of the potential role of PLA2 in stimulus-secretion coupling, we examined the relationship between PLA2 activation and the surface expression of CD11b/CD18 (MAC-1) in human polymorphonuclear leukocytes (hPMNL), including the functional consequences of PLA2 inactivation on MAC-1-dependent adhesion. The selective inhibition of PLA2 by the marine natural products manoalide (MLD) and scalaradial (SLD) blocks [3H]arachidonic acid (AA) release in calcium ionophore A23187-stimulated neutrophils, and also inhibits secretion of specific and azurophilic granule constituents. Additional studies demonstrate that MLD, SLD, and other less potent PLA2 inhibitors such as 4-bromophenacylbromide and nordihydroguiaretic acid inhibit the surface expression of MAC-1 (IC50: MLD, 0.33 microM; SLD, 0.23 microM; 4-bromophenacylbromide, 2.8 microM; NDGA, 3.5 microM) at concentrations similar to those at which they inhibit [3H]AA release. Inhibitors of cyclooxygenase, 5-lipoxygenase, protein kinase C, or calcium channel antagonists have no effect on MAC-1 expression. PLA2 inactivation also prevents MAC-1 up-regulation in hPMNL stimulated with FMLP, IL-8, TNF-alpha, PMA, or platelet activating factor. In FMLP-stimulated hPMNL, under conditions in which no secondary granule constituents are secreted, MAC-1 and alkaline phosphatase up-regulation from intracellular granules is inhibited by MLD and SLD. Functional assays also demonstrate that MLD and SLD block MAC-1-dependent adhesion of activated neutrophils to keyhole limpet hemocyanin at concentrations that block the surface expression of MAC-1. [3H]AA release and MAC-1 expression in MLD and SLD-treated hPMNL could be recovered in the presence of 1 mM hydroxylamine in a time-dependent fashion, consistent with reported data that MLD and SLD inactivate PLA2 through Schiff base formation. In summary, these data emphasize the role of PLA2 as a key regulator of MAC-1 expression in models of neutrophil adhesion.
Triggering of degranulation in mast cells by exogenous type II phospholipase A2.
Authors: Murakami M , Hara N , Kudo I , Inoue K
Published: December 2022

We have previously shown the possibility that endogenous type II phospholipase A2 (PLA2) might participate in degranulation in mast cells (MC) (Murakami, M., et al. 1992. Eur. J. Biochem. 209:257). Now we have examined whether or not exogenously added type II PLA2 triggers MC degranulation. When rat peritoneal connective tissue MC (CTMC) were exposed to purified rat type II PLA2 at concentrations of more than 10 micrograms/ml, significant release of histamine was observed, whereas PGD2 was not generated under the same conditions. Mouse peritoneal CTMC as well as bone marrow-derived immature MC also responded to PLA2. Preincubation of CTMC with tyrosine kinase inhibitors, genistein, and herbimycin A, but not with pertussis toxin, resulted in abolition of the sensitivity to PLA2. The ability of type II PLA2 to induce histamine release was inhibited by an antibody or chemicals, both of which blocked the catalytic activity of type II PLA2. Heparin or an antibody recognizing the heparin-binding domain of type II PLA2 also suppressed the MC-degranulating activity, probably due to inhibition of binding of PLA2 to the cells. The interaction between heparan sulfate on cell surface and the heparin-binding domain of type II PLA2 may be important for the induction of exocytosis. The catalytic domain of the enzyme is also crucially important for the degranulation induction. Furthermore, we found that nerve growth factor, one of the potent regulators of MC function, significantly potentiated type II PLA2-induced histamine release from rat CTMC. These results suggest the possible role of extracellular type II PLA2 in activation of CTMC primed with nerve growth factor at inflamed sites.
Murine recipients of fully mismatched donor marrow are protected from lethal graft-versus-host disease by the in vivo administration of rapamycin but develop an autoimmune-like syndrome.
Authors: Blazar B R , Taylor P A , Snover D C , Sehgal S N , Vallera D A
Published: December 2022

We investigated the ability of the macrolide antifungal agent rapamycin (RAPA) to inhibit murine graft-vs-host disease induced across the MHC barrier. An optimum dose (1.5 mg/kg) given for 14 days beginning on the day of transplant (and then three times weekly until 1 mo) effectively and significantly (p < 0.001) protected 80% of irradiated B10.BR recipients of C57Bl/6 bone marrow/spleen grafts for over 90 days, whereas 80% of control mice died by day 37. Using a congenic model in which a mixture of Ly5.1+ bone marrow (T cell-depleted) and Ly5.2+ spleen cells allowed us to distinguish mature and immature cells, we found that RAPA inhibits the splenic expansion of mature donor-derived T cells in B10.BR recipients after bone marrow transplantation. In addition, phenotyping studies revealed that RAPA causes a massive reduction of immature CD4+CD8+ T cells in the thymus, indicating that RAPA probably interferes with maturation of immature CD3-CD4-CD8- T cells to CD4+CD8+ T cells. There was also a predilection toward development or intrathymic retention of the more mature CD3+CD4-CD8+ or CD3+CD4+CD8- cells in the thymus of long term survivors. These same observations were made in different experiments with mice given syngeneic bone marrow transplantation and RAPA. However, RAPA administration was associated with the occurrence of an autoimmune-like syndrome, consisting of ulcerative dermatitis, hepatic bile duct proliferation, and nondestructive lymphoid peribronchiolar infiltration of the lung. RAPA interfered with the deletion of potentially self-reactive T cells that occurs in thymic development. The failure of clonal deletion was observed in allogeneic and syngeneic transplants given RAPA, although only the allografted mice experienced an autoimmune-like syndrome. Some, but not all, of the nondeleted V beta populations were functionally active. These new findings bear certain dissimilarities to the syndrome and lesions observed with cyclosporin A treatment, particularly in the observation of bile duct proliferation and ulcerative skin lesions. Nonetheless, because of the potent effect of RAPA in preventing lethal graft-vs-host induced across the MHC, further investigation of the immune consequences of this highly effective compound is warranted.
Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.
Authors: Giudice G J , Emery D J , Zelickson B D , Anhalt G J , Liu Z , Diaz L A
Published: December 2022

Bullous pemphigoid (BP) and herpes gestationis (HG) are skin diseases characterized by subepidermal blisters and autoantibodies against two hemidesmosomal Ag, i.e., BP230 and BP180. Based on sequence analysis the BP180 Ag was predicted to be a transmembrane protein with a long extracellular collagenous domain. In the present investigation fusion proteins encompassing various segments of the BP180 Ag were expressed in a prokaryotic system and assayed by immunoblotting and immunoadsorption against a panel of BP, HG and control sera. One antigenic site, comprising 14 amino acids of the BP180 noncollagenous (NC) 16A domain, was shown to be recognized by 60% of BP sera and by 63% of HG sera tested. 73% (11/15) of BP sera and 100% (8/8) of HG sera reacted with at least one of three BP180 fusion proteins representing various portions of the NC16A domain. Immunoadsorption analysis identified this region of BP180 as an immunodominant site. Using an affinity purified rabbit antiserum raised against a recombinant form of BP180, this BP/HG autoantibody-reactive region was localized to the epidermal basal lamina immediately adjacent to the hemidesmosome. These findings confirmed the predicted type II transmembrane orientation of the BP180 Ag. Thus, the long, C-terminal collagenous domain of this basal keratinocyte protein projects into the basal lamina and may function as a site of interaction with an extracellular matrix component. It is proposed that autoantibodies directed against the well-defined antigenic site on the BP180 ectodomain may play an initiatory role in subepidermal blister formation in BP and HG patients.
Clonal predominance of T cell receptors within the CD8+ CD45RO+ subset in normal human subjects.
Authors: Hingorani R , Choi I H , Akolkar P , Gulwani-Akolkar B , Pergolizzi R , Silver J , Gregersen P K
Published: December 2022

Structural models for the TCR alpha/beta predict that the CDR1, CDR2, and CDR3 loops of both the alpha- and beta-chains contribute to specific interactions with the Ag/MHC complex. The CDR3 loops are constructed by joining events involving the V-(D)-J segments, and thus may vary in both sequence and length. We have developed a polymerase chain reaction assay to assess the length variation of the CDR3 loop in TCR derived from seven V beta segment families (V beta 2, V beta 3, V beta 4, V beta 9, V beta 14, V beta 16, and V beta 17). Peripheral blood T cells from 10 normal adults as well as five cord blood samples were studied. CD4+ and CD8+ T cells were analyzed separately. We observed extreme predominance of particular CDR3 lengths in half of the normal adults. These TCR were shown to be clonal by direct sequence analysis. This clonal dominance was found in the CD8+, CD45RO+ T cell population, and was observed in various V segment families. These patterns of TCR clonality were persistent over many months of observation in some individuals. In one subject, the new appearance of a predominant clone was associated with a booster vaccination for hepatitis B. These studies reveal a surprising degree of oligoclonality in the CD8+ cells of normal subjects which may be due to both environmental and genetic factors; the functional significance of persistent clonal dominance in the CD8 compartment remains to be determined.
Exacerbation of murine herpes simplex virus-mediated stromal keratitis by Th2 type T cells.
Authors: Jayaraman S , Heiligenhaus A , Rodriguez A , Soukiasian S , Dorf M E , Foster C S
Published: December 2022

Corneal infection of susceptible mice with HSV-1 causes herpetic stromal keratitis (HSK), which serves as a model of human HSK. To study the properties of the T lymphocytes involved in HSK, susceptible mice were immunized with the synthetic peptide corresponding to the amino terminal of HSV-1-associated glycoprotein D (gD 5-23). A CD4+ long-term T cell line and a clone bearing V beta 8.2 TCR were derived from peptide-primed lymph node cells. These T cells recognize gD 5-23 peptide in the context of I-Ed and require CD4 and LFA-1 for Ag-specific proliferation. Significantly, a truncated peptide gD 15-23 induced vigorous proliferation, indicating that these 9 amino acids constitute an epitope recognized by these T cells. The gD-specific T cells produced IL-4 and used it as the autocrine growth factor and hence belong to the Th2 subtype. Adoptive transfer of gD-specific Th2 cells into susceptible mice increased both the onset and severity of HSK after corneal HSV-1 infection. Injection of gD-specific Th2 cells without HSV-1 infection failed to cause eye damage. In addition, an irrelevant Ag-specific Th2 clone failed to induce similar tissue damage when the corresponding Ag was applied to the eye. These data indicate that the T cell-mediated exacerbation of HSK in these studies is dependent on the specific recognition of gD after corneal HSV-1 infection. Finally, gD-specific Th2 cell transfer also rendered HSK-resistant mice susceptible for HSK, suggesting that the freedom from HSK in resistant mice may primarily be due to their inability to produce the pathogenic Th2 cells. The data collectively implicate an important role for Th2 cells in the induction of HSV-mediated keratitis in mice.
T cell peptide of a self-protein elicits autoantibody to the protein antigen. Implications for specificity and pathogenetic role of antibody in autoimmunity.
Authors: Lou Y , Tung K S
Published: December 2022

A 13-mer peptide of the ZP3 glycoprotein from mouse zona pellucida has a T cell epitope that induces autoimmune oophoritis and a B cell epitope that reacts with antibody to murine ZP3. When the B cell epitope was partially truncated, the ZP3 peptides no longer induced antibody to the B cell epitope, but unexpectedly they elicited antibody to the zona pellucida. These autoantibodies were of IgG class, detected in sera and bound to the ovarian zona pellucida. That an exclusive T cell peptide of murine ZP3, without coinjection of the whole ZP3 protein, elicited autoantibodies against ZP3 outside the T cell peptide was confirmed as follows. First, the ZP3 T cell peptide did not contain additional B cell epitopes that cross-reacted with native ZP3. Second, endogenous ovarian Ag were required because autoantibodies were not detected in ovariectomized mice immunized with ZP3 peptides lacking the B epitope. This autoantibody amplification phenomenon demonstrates conclusively that 1) self-reactive B cells for ovarian autoantigens respond to endogenous ovarian Ag in vivo after activation of ZP3-specific Th cells and 2) serum antibody in an autoimmune disease need not mirror the immunogen that initiates the disease process. Nonetheless, the autoantibodies bound to the zona pellucida in vivo and are potentially important in disease pathogenesis.
Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP85/CD44)-cytoskeleton interaction.
Authors: Bourguignon L Y , Lokeshwar V B , Chen X , Kerrick W G
Published: December 2022

The purposes of this study are to characterize the binding of hyaluronic acid (HA) to mouse T lymphoma cells, to measure changes in intracellular Ca2+ after HA binding, to elucidate the interaction between the HA receptor, GP85(CD44), and ankyrin in the membrane skeleton, and finally to correlate these events with HA receptor patching/capping and cell adhesion to HA. First, we established an in vivo assay using [3H]HA to measure the binding of HA to mouse T lymphoma cells, and found that the binding of [3H]HA to these cells is readily inhibited by the addition of anti-GP85(CD44) antibody suggesting that GP85(CD44) is the HA receptor. Next, we examined various signal transducing events that occur after HA binds to its receptor on mouse T lymphoma cells. The results of these studies indicate that the concentration of intracellular Ca2+ (as measured by Fura-2 fluorescence) begins to increase within seconds, and reaches a maximal level 5 min after the addition of HA to the cells. After this increase of intracellular Ca2+, HA induces both its receptors, GP85(CD44), to form patched/capped structures, and cell adhesion to HA-coated plates. Furthermore, we have determined that GP85(CD44) binds directly and specifically to ankyrin (Kd approximately 1.94 nM) in a saturable manner; and that ankyrin is preferentially accumulated underneath the HA-induced GP85(CD44) capped structures. The Ca2+ ionophore, ionomycin, was found to stimulate HA-induced receptor capping and adhesion while EGTA (a Ca2+ chelator), nefedipine/bepridil (Ca2+ channel blockers), W-7 (a calmodulin antagonist), and cytochalasin D (a microfilament inhibitor), but not colchicine (a microtubule disrupting agent), inhibit HA-induced receptor redistribution and adhesion to HA-coated plates. These findings strongly suggest that ankyrin plays an important role in linking the HA receptor, GP85(CD44), to the membrane-associated actomyosin contractile system during hyaluronic acid-mediated lymphocyte activation.
alpha beta T cell receptor-positive intraepithelial lymphocytes with CD4+, CD8- and CD4+, CD8+ phenotypes from orally immunized mice provide Th2-like function for B cell responses.
Authors: Fujihashi K , Yamamoto M , McGhee J R , Kiyono H
Published: December 2022

Our previous studies have shown that murine alpha beta TCR+ intraepithelial lymphocytes (IEL) contained T cells that can provide B cell help. In this study, we have examined the three subsets of alpha beta TCR+ IEL for Ag-specific helper function and cytokine production because alpha beta TCR+ IEL are divisible into three subsets including CD4+, CD8- T cells, CD4+, CD8+ double positive (DP) T cells, and CD4-, CD8+ T cells. When these three subsets of alpha beta TCR+ IEL from C3H/HeN mice (H-2k) orally immunized with SRBC were cultured with splenic B cells, adherent cells, and SRBC, both CD4+, CD8- and DP T cell fractions supported IgM, IgG1, and IgA anti-SRBC responses, whereas the CD4-, CD8+ T cell subset did not exhibit helper function. Addition of anti-I-Ak mAb resulted in the reduction of SRBC-specific PFC responses, whereas anti-H-2Kk mAb did not affect the CD4+, CD8- and DP T cell-supported B cell responses. Furthermore, when these CD4-bearing T cells from mice orally immunized with SRBC were co-cultured with B cells and adherent cells in the presence of unrelated Ag (e.g., horse RBC), SRBC-specific B cell responses were not induced. When type 1 and type 2 Th cell cytokine production was examined by IFN-gamma, IL-2-, IL-4-, and IL-5-specific enzyme-linked immunospot assays, increased numbers of IL-4- and IL-5-secreting type 2 Th cells, whereas lower numbers of IFN-gamma and IL-2-producing type 1 Th cells were seen in CD4+, CD8- and DP T cell fractions on in vitro stimulation with SRBC. In the case of CD4-, CD8+ T cells, approximately equal and low numbers of type 2 cytokine-producing cells were noted in both SRBC-stimulated and -unstimulated cultures. Furthermore, when different fractions of IEL T cells from Ag-stimulated and -unstimulated cultures were characterized for cytokine-specific mRNA by reverse transcription polymerase chain reaction, stronger bands for IL-4 and IL-5 were detected in both CD4+, CD8- and DP IEL subsets when compared with the unstimulated cells. In contrast, the change in intensity of the band of polymerase chain reaction product for IFN-gamma was slight or unchanged in Ag-stimulated CD4-bearing IEL when compared with unstimulated cells.(ABSTRACT TRUNCATED AT 400 WORDS)
IFN-gamma and lipopolysaccharide differentially modulate expression of tumor necrosis factor receptor mRNA in murine peritoneal macrophages.
Authors: Tannenbaum C S , Major J A , Hamilton T A
Published: December 2022

Expression of TNF receptor (TNFR) mRNA has been examined in murine peritoneal macrophages stimulated with LPS and/or IFN-gamma. LPS markedly enhanced expression of a heterogenous population of mRNA, which hybridized with a cDNA encoding the type II TNFR. mRNA expression was optimally induced by 4 to 8 h and returned to baseline by 24 h after stimulation. Interestingly, though IFN-gamma can synergize with LPS for the expression of TNF-alpha, it abrogated the LPS-mediated enhancement of type II TNFR in a dose-dependent fashion. IFN-alpha, though less effective, had a qualitatively comparable effect. These effects were selective for the type II TNFR because levels of mRNA encoding the type I TNFR did not vary appreciably with any of the treatments described. The effects of IFN-gamma on LPS-mediated TNFR expression were dependent on the sequence of exposure; pretreatment with IFN-gamma was most effective at blocking response to LPS, whereas IFN-gamma added 1 h after initiation of LPS treatment had little or no effect. The effects of both LPS and IFN-gamma on type II TNFR expression were mediated at least in part by modulation of transcription. The effects of both LPS and IFN-gamma were also independent of protein synthesis because inclusion of cycloheximide in the treatment protocol did not abrogate either the inductive or the suppressive effects. These findings suggest that IFN-gamma and LPS modulate the physiologic action of TNF through complex mechanisms involving effects on the transcription of TNF-alpha itself and on receptors through which it may act in autocrine or paracrine fashion.
Polymorphism in BoLA-DRB3 exon 2 correlates with resistance to persistent lymphocytosis caused by bovine leukemia virus.
Authors: Xu A , van Eijk M J , Park C , Lewin H A
Published: December 2022

The relationship between polymorphism of the bovine lymphocyte Ag (BoLA)-DRB3 gene and resistance and susceptibility to persistent lymphocytosis (PL) caused by bovine leukemia virus (BLV) was investigated. Exon 2 of the BoLA-DRB3 gene was cloned from animals with BoLA haplotypes previously found to be associated with resistance and susceptibility to PL. Sequence analysis revealed the presence of the amino acids Glu-Arg (ER) at putative Ag binding residues 70 and 71 only in BoLA haplotypes associated with resistance to PL. This correlation was confirmed in a case control study (n = 26) using an allele-specific polymerase chain reaction for the detection of ER at residues 70-71. These results provide a molecular basis for Ir gene control of resistance and susceptibility to PL and suggest that the cellular immune response is important in preventing the in vivo spread of BLV infection.
Kinetic analysis of antigen-specific immune responses in resistant and susceptible mice during infection with Trypanosoma cruzi.
Authors: Hoft D F , Lynch R G , Kirchhoff L V
Published: December 2022

The Tulahuén strain of Trypanosoma cruzi has been shown previously to cause higher parasitemias and greater mortality in BALB/c mice compared with C57BL6/J mice. The goal of our study was to determine whether different cytokine responses to parasite Ag during T. cruzi infection correlate with the susceptible and resistant phenotypes identified in these mice. At several time points after initial infection with insect-derived metacyclic trypomastigotes, lymph node and spleen cell suspensions were prepared from animals of each mouse strain. These lymphocyte suspensions were stimulated with Ag prepared from cultured parasites and the production of IFN-gamma, IL-4, IL-2, and IL-5 was measured. Lymphocytes from C57BL6/J mice produced 10-fold more IFN-gamma than BALB/c lymphocytes. However, this enhanced response occurred only for a limited time preceding peak parasitemias. Ag-induced secretion of IL-4 from BALB/c lymphocytes was detectable by 2 wk of infection and increased during the 2nd and 3rd mo of infection. Most C57BL6/J culture supernatants did not contain measurable levels of IL-4. Lymphocytes from both murine strains produced levels of IL-2 and IL-5 indistinguishable from uninfected controls. These results indicate that increased numbers or potency of lymphocytes that produce Ag-specific IFN-gamma responses are present in resistant mice during T. cruzi infection. This phenomenon may be responsible for the lower parasitemias seen in C57BL6/J mice. However, even these relatively resistant mice become chronically infected with T. cruzi, and spleen cells from infected mice can suppress IFN-gamma induced by heterologous Ag. Our data suggest that IL-4 production is a marker for the T. cruzi susceptible phenotype. Differential production of IL-2 or IL-5 was not found, suggesting that these cytokines are not important factors in T. cruzi resistance or susceptibility.
Human granzyme B degrades aggrecan proteoglycan in matrix synthesized by chondrocytes.
Authors: Froelich C J , Zhang X , Turbov J , Hudig D , Winkler U , Hanna W L
Published: December 2022

Cartilage degradation, a hallmark of rheumatoid arthritis, is attributed to serine and metalloproteases secreted by neutrophils, synovial lining cells, macrophages, and chondrocytes. A large proportion of synovial fluid lymphocytes contains the granule-associated serine proteases granzymes A and B. We report that lysates of IL-2-stimulated lymphocytes contain an enzymatic activity (ECMase; cartilage extracellular matrix 35S release assay; extracellular matrix degrading activity) that solubilizes matrix synthesized by chondrocyte monolayers. ECMase activity is inactivated by the serine protease inhibitor diisopropylfluorophosphate, is stored in dense granules and cleaves aggrecan proteoglycans but not free glycosaminoglycans, hyaluronic acid, or type II collagen. ECMase is mediated by a cationic protein with biochemical properties identical to granzyme B, inasmuch as it preferentially hydrolyzes the substrate Boc-Ala-Ala-Asp-SBzl, immunochemically cross-reacts with an antibody that binds to a conserved amino-terminal region of lymphoid-myeloid serine proteases, and has amino-terminal sequence identity with human Q31 granzyme B. Using an agarose gel electrophoresis technique to assess cleavage of the rat sarcoma aggrecan, the catalytic efficiency of granzyme B for the digestion of aggrecan (catalytic efficiency = 1.7 x 10(7) M-1 s-1) was 425-fold faster than the catalytic efficiency reported for human stromelysin-1 at pH 7.5 (catalytic efficiency 4000 M-1 s-1) and 3200-fold faster than granzyme A. Based on these observations, we propose that granzyme B, secreted from cytotoxic lymphocytes within the rheumatoid joint, may contribute to cartilage loss by degrading resident aggrecan.
Comparison of the allergenicity of ovalbumin and ovalbumin peptide 323-339. Differential expansion of V beta-expressing T cell populations.
Authors: Renz H , Bradley K , Larsen G L , McCall C , Gelfand E W
Published: December 2022

We analyzed the effects of sensitization of BALB/c mice to the OVA peptide amino acids 323-339, on the development of an IgE response, immediate cutaneous hypersensitivity and airways responsiveness (AR). Daily aerosolization of OVA 323-339 for 20 min over a period of 10 days was as effective in the stimulation of a serum anti-OVA IgE antibody response as sensitization to native OVA by the same route. After sensitization to native OVA, the majority of the IgE anti-OVA response was directed against peptide 323-339. The antibody responses were paralleled by skin test responses in sensitized mice: 73% of OVA-sensitized mice developed immediate type reactions when tested with native OVA and 82% of the mice had positive immediate skin test responses to intradermal injection of peptide 323-339. After sensitization to the peptide, 69% of the mice had positive responses to native OVA and 77% responded to peptide 323-339. Aerosolization of OVA as well as OVA 323-339 led to a comparable increase in airway responsiveness as measured by electrical field stimulation of tracheal smooth muscle preparations. To characterize T cell subpopulations that were stimulated after allergen sensitization, the distribution of specific V beta-expressing T cells was analyzed in local draining lymph nodes of the airways and the lungs. These lymph nodes were found to be enlarged after both OVA and OVA peptide sensitization. Sensitization to native OVA resulted in an increased percentage of V beta 8.1 and V beta 8.2 T cells whereas selective stimulation of V beta 8.1 T cells was found after peptide sensitization. These data indicate that OVA peptide 323-339 represents a T and B cell epitope of OVA, which is important in the generation and development of immediate hypersensitivity responses in BALB/c mice.
Induction of lupus-associated autoantibodies by immunization with native and recombinant Ig polypeptides expressing a cross-reactive idiotype 4B4.
Authors: Dang H , Ogawa N , Takei M , Lazaridis K , Talal N
Published: December 2022

A human mAb designated 4B4 with anti-Sm activity was derived from a patient with systemic lupus erythematosus. This antibody expressed a lupus-associated cross-reactive Id, partially related to the monoclonal murine anti-Sm (Y2) from MRL/lpr mice. Studies were performed to investigate the ability of 4B4 to induce lupus in nonautoimmune-prone mice. BALB/c mice immunized with 4B4 produced antibodies to dsDNA, ssDNA, Sm ribonucleoprotein, and mouse Fc fragment. There was no antibody activity against SSA/Ro, SSB/La, and hen egg lysozyme. Ag inhibition studies show that the autoantibodies were not polyreactive. Mice were also immunized with r4B4 polypeptides representing the H/L heterodimer, H chain and L chain. Autoantibodies were induced in mice immunized against the H/L and H polypeptides. No autoantibodies were induced in mice immunized with recombinant L chain. Furthermore, from 20 to 68% of antibody activity to Sm or dsDNA could be inhibited with anti-Id antiserum (either anti-4B4 or Y2). The autoantibody was initially IgM and then underwent an isotype switch to IgG. These results show that lupus-associated autoantibodies can be induced by immunization with 4B4 and that the 4B4 VH region is important in this induction process. The finding of murine IgG autoantibody expressing a cross-reactive Id similar to the immunizing 4B4 suggests a role for anti-idiotypic Th cells in this autoimmune response.
Novel T cell antigen 4-1BB associates with the protein tyrosine kinase p56lck1.
Authors: Kim Y J , Pollok K E , Zhou Z , Shaw A , Bohlen J B , Fraser M , Kwon B S
Published: December 2022

4-1BB is a 30-kDa inducible T cell Ag, and is expressed predominantly as a 55-kDa dimer on both CD4+ and CD8+ T lymphocytes. The cytoplasmic tail of 4-1BB contains the sequence Cys-Arg-Cys-Pro, which is similar to the sequence Cys-X-Cys-Pro, which mediates the binding of the CD4 and CD8 molecules to the protein tyrosine kinase p56lck. An anti-4-1BB mAb, 53A2, was used to determine whether 4-1BB may associate with p56lck. The 53A2 mAb specifically recognized 4-1BB on a CD8+ T cell line, CTLL-2, and coimmunoprecipitated a 56-kDa protein along with 4-1BB. Peptide mapping indicated that the 56-kDa phosphoprotein was identical to p56lck. The coimmunoprecipitation of p56lck with 4-1BB also occurred in nonlymphoid cells such as insect (Sf-21) and HeLa cells when the two recombinant proteins were coexpressed. Analysis of mutant p56lck recombinant proteins showed that two cysteine residues critical for p56lck-CD4 (or -CD8) complex formation are also required for the p56lck-4-1BB interaction. These studies establish that 4-1BB physically associates with p56lck.
IL-10 and viral IL-10 prevent IL-4-induced IgE synthesis by inhibiting the accessory cell function of monocytes.
Authors: Punnonen J , de Waal Malefyt R , van Vlasselaer P , Gauchat J F , de Vries J E
Published: December 2022

In the present study it is demonstrated that IL-10 and viral (v)-IL-10 inhibit IL-4-induced IgG4 and IgE synthesis in cultures of PBMC from healthy nonatopic donors. The inhibition occurred at the mRNA level. IL-10 strongly reduced IL-4-induced expression of both germline and productive epsilon transcripts in PBMC. The inhibitory effects were completely neutralized, or IgG4 and IgE synthesis was even enhanced, by anti-IL-10 mAb, demonstrating the specificity of the reaction. IL-4-induced IgG4 and IgE synthesis by PBMC was monocyte dependent. IL-4 failed to induce IgG4 or IgE synthesis by monocyte-depleted PBMC, but the production of these isotypes was completely restored by reconstitution with autologous monocytes. However, monocytes preincubated with IL-10 for 24 h failed to provide the accessory signals required for IL-4-induced IgG4 and IgE synthesis, indicating that the inhibitory effects of IL-10 are indirectly mediated via monocytes. This notion was further supported by the finding that IL-10 and v-IL-10 failed to inhibit IL-4-induced IgE synthesis in the absence of monocytes, i.e., when highly purified B cells were cultured in the presence of anti-CD40 mAb or cloned activated CD4+ T cells. Moreover, IL-10 failed to inhibit IL-4-induced germline epsilon mRNA synthesis in highly purified B cells. The inhibitory effects of IL-10 could not be restored by exogenous TNF-alpha or IL-6, indicating that the inhibitory effects were not mediated through inhibition of production of these cytokines. This is compatible with the observation that monocytes preincubated with IL-10 did not inhibit IgG4 or IgE synthesis in a monocyte- and T cell-independent culture system, in which purified B cells were cultured in the presence of anti-CD40 mAb and IL-4. Collectively, these data indicate that IL-10 mediates a potent, monocyte-dependent, inhibitory effect on IL-4-induced IgG4 and IgE production by human PBMC.
Monocyte-regulated IFN-gamma production in human T cells involves CD2 signaling.
Authors: Wingren A G , Dahlenborg K , Björklund M , Hedlund G , Kalland T , Sjögren H O , Ljungdahl A , Olsson T , Ekre H P , Sansom D
Published: December 2022

Cooperation between monocytes and T lymphocytes is essential for several aspects of immunologic activation. We have utilized PHA and IL-2-activated human T cells to characterize the role of monocytes in the regulation of T cell-derived IFN-gamma production. The limited IFN-gamma production by isolated T cells in this culture system was increased more than 10-fold when monocytes were added. No influence of monocytes was observed on TNF production or T cell proliferation. Maximal level of IFN-gamma in the cell culture supernatants was obtained when monocytes were added within 12 h after activation of the T cells with IL-2 and PHA. Addition of monocytes 48 h after activation resulted in marginal production of IFN-gamma, suggesting that T cells are sensitive to the monocyte-related signal during a short time period after activation. Cell-to-cell contact between the T cells and accessory cells was found to be necessary for enhanced IFN-gamma production because separation of the cells with a semipermeable membrane abolished the effect. mAb blocking experiments suggested the involvement of the CD2/LFA-3 but not the LFA-1/ICAM-1 pathway in monocyte regulation of T cell synthesis of IFN-gamma. Chinese hamster ovary (CHO) cells transfected with LFA-3 (CHO-LFA-3) and HLA-DR4/LFA-3 (CHO-DR4/LFA-3) strongly enhanced T cell IFN-gamma production, whereas untransfected CHO cells, CHO cells transfected with ICAM-1 (CHO-DR4/ICAM-1), and HLA-DR4 (CHO-DR4) did not support IFN-gamma production. PCR analysis and in situ hybridization demonstrated enhanced IFN-gamma mRNA levels in T cells stimulated in the presence of CHO-DR4/LFA-3 compared with untransfected CHO cells, indicating that the CD2/LFA-3 pathway regulates IFN-gamma production at the mRNA level. CHO-LFA-3 and CHO-DR4/ICAM-1 cells mediated strong adhesion to T cells, whereas untransfected CHO cells and CHO-DR4 cells failed to mediate adhesion. This suggests that the ability of CHO-LFA-3 but not CHO-DR4/ICAM-1 cells to induce IFN-gamma production was attributed to signal transduction rather than cell adhesion only.
A novel enhancer element in the human IL-4 promoter is suppressed by a position-independent silencer.
Authors: Li-Weber M , Krafft H , Krammer P H
Published: December 2022

IL-4 is a pleiotropic cytokine that regulates T cell-dependent immune responses. We identified and characterized a novel enhancer, positive regulatory element I (PRE-I), in the 5' region of the promoter of the human IL-4 gene. The functional core element of PRE-I is -239GTGTAATTTCCTATGC-224. Two novel nuclear proteins, POS-1 and POS-2, were found to specifically bind to the core element and function as transcriptional activators. The function of PRE-I did not require T cell stimulation and was not restricted to T cells. However, mutations in the core element of PRE-I significantly reduced the promoter activity and completely impaired inducibility of the promoter. In the human IL-4 promoter the enhancer function of PRE-I is strongly suppressed. A negative regulatory element (NRE), 45 bp upstream of PRE-I, directly represses PRE-I enhancer activity. Repression of PRE-I by NRE does not require a particular order or arrangement of positive and negative regulatory sequences. The IL-4 NRE may also down-regulate other enhancers, such as the murine sarcoma virus and the SV40 enhancers. Thus, the IL-4 NRE may represent a new type of cis regulatory element that carries the properties of a silencer but down-regulates enhancer instead of basal promoter activity.
Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists.
Authors: Mohler K M , Torrance D S , Smith C A , Goodwin R G , Stremler K E , Fung V P , Madani H , Widmer M B
Published: December 2022

Two forms (monomeric or dimeric) of the extracellular, ligand-binding portion of the human p80 cell-surface receptor for TNF were used to antagonize TNF activity in vitro and in vivo. The dimeric sTNFR:Fc molecule was a more potent inhibitor of TNF than the monomeric sTNFR (50 to 1000x), as assessed in vitro by inhibition of TNF binding or bioactivity and in vivo by protection of mice from an otherwise lethal injection of LPS. Surprisingly, the dimeric sTNFR:Fc construct demonstrated a beneficial effect even when administered 3 h after a lethal LPS injection (i.e., after serum TNF levels had peaked and receded). To study the mechanism by which the soluble TNFR functions in vivo, serum TNF levels were examined in mice given LPS in the presence or absence of soluble receptor. Administration of a mortality-reducing dose of sTNFR:Fc ablated the rise in serum TNF bioactivity that normally occurs in response to LPS. However, TNF bioactivity was revealed in these "TNF-negative" serum samples when the L929 bioassay was modified by inclusion of a mAb that blocks the binding of murine TNF to the human soluble TNFReceptor. These results indicate that the absence of direct cytolytic activity in the L929 assay was caused by neutralization of TNF, rather than to an absence of TNF in the serum. Moreover, administration of either monomeric sTNFR or low doses of dimeric sTNFR:Fc actually resulted in increased serum TNF levels compared to mice given LPS but no soluble receptor. However, these "agonistic" doses of soluble receptor did not lead to increased mortality when an LD60 dose of LPS was given. Thus, dimeric sTNFR are effective inhibitors of TNF and under some circumstances function simultaneously as both TNF "carriers" and antagonists of TNF biologic activity.
A hit-and-run system for introducing mutations into the Ig H chain locus of hybridoma cells by homologous recombination.
Authors: Bautista D , Shulman M J
Published: December 2022

The traditional method of site-specific mutagenesis is to introduce predetermined mutations into an expression vector, which is then transferred to cells so that the relevant gene product and the effects of the mutations can be measured. A problem with this approach is that the expression of the transferred genes varies from transformant to transformant, presumably because the number of vector copies and the site of chromosomal integration vary among transformants. While it should be possible to avoid this variability by mutagenizing the chromosomal gene itself, the labor involved in introducing predetermined mutations by homologous recombination with a mutagenized vector is usually so intense that this has not been the favored method. We describe here a system for introducing mutations into the IgH locus of hybridoma cells. This system greatly reduces the labor that would usually be required to identify and recover the rare recombinants. This is a two-step, so-called "hit-and-run," method, whereby mutations are first introduced into the chromosomal locus by targeted vector integration, after which the vector is excised so as to leave the mutation in the chromosomal target. The first step employs an enhancer trap vector bearing an enhancerless gpt gene; using this vector the frequency of randomly inserted transformants which grow in mycophenolic acid containing selective medium is so low that approximately 25% of the selected transformants have integrated the vector into the IgH locus by homologous recombination. Properly targeted transformants can then be used to derive secondary recombinants that have excised the vector and thus become gpt-. This second step which involves selection of gpt- cells by their resistance to 6-thioxanthine is also efficient, in that approximately 75% of the treated cells have excised the gpt gene by homologous recombination. Overall the labor involved in mutagenizing the chromosomal locus is not much more than is needed to produce the traditional transformants expressing a mutagenized transferred gene.