bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell recognition of transforming proteins encoded by mutated ras proto-oncogenes.
Authors: Peace D J , Chen W , Nelson H , Cheever M A
Published: December 2022

Activated ras proto-oncogenes contribute to the pathogenesis of many animal and human malignancies. ras proto-oncogenes are generally activated by point mutations within codons 12 or 61, which result in the expression of ras protein (p21) bearing characteristic single amino acid substitutions at the corresponding residues. The purpose of the current study was to determine whether the presence of single transforming amino acid substitutions can render normal ras protein immunogenic and, thus, a possible target for T cell-mediated tumor therapy. In initial experiments, C57BL/6 mice were immunized with a synthetic peptide corresponding to residues 5 through 16 of p21 containing the transforming substitution of arginine for normal glycine at residue 12. The results demonstrated that class II MHC-restricted T cells which were specific for the peptide could be elicited, and that the peptide-induced T cells could specifically recognize the corresponding intact p21 ras protein. Recognition of p21 ras protein by peptide-specific T cells implies that C57BL/6 APC can process the activated ras protein in a fashion that allows presentation of digested protein by class II MHC molecules in a configuration similar to the configuration with synthetic peptide. Evaluation of the immunogenicity of peptides containing alternative transforming amino acid substitutions of ras protein demonstrated that some, but not all, were immunogenic in individual strains of mice. Therefore, although ras protein-specific T cells can be elicited by immunization with synthetic peptides, not all of the potential ras mutations commonly associated with malignancy may be recognizable by T cells from all individuals.
Substance P acts directly upon cloned B lymphoma cells to enhance IgA and IgM production.
Authors: Pascual D W , Xu-Amano J C , Kiyono H , McGhee J R , Bost K L
Published: December 2022

The IgA producing murine B lymphoma, CH12.LX.C4.4F10 (4F10) and the IgM producing murine lymphoma, CH12.LX.C4.5F5 (5F5) were found to express substantial numbers of substance P (SP) receptors having dissociation constants equal to 0.69 nM. Binding of SP by these B lymphoma cells was via the tachykinin-specific C-terminus sequence, Phe-X-Gly-Leu-Met-NH2, because SP, SP antagonist (D-Pro2-D-Phe7-D-Trp9-SP), eledoisin, and substance K could effectively inhibit radiolabeled SP binding, whereas the SP N-terminus fragment, SP (1-4), could not. The functionality of these receptors could be demonstrated by the ability of subnanomolar concentrations of SP to induce Ig secretion in a dose-dependent fashion. However, the presence of a second stimulus in these cultures was required to obtain maximal increases. IgA secretion by 4F10 cells was elevated only 25 to 37%, and IgM secretion by 5F5 cells was not significantly increased in cultures in which nanomolar concentrations of SP were present. Conversely, coculturing 5F5 cells with a suboptimal concentration of LPS (50 ng/ml) and 10(-10)M SP resulted in an approximate threefold increase in supernatant IgM when compared to control cultures stimulated with LPS alone. While not as dramatic, 10(-10) M SP also enhanced IgA secretion of LPS-stimulated 4F10 cells by approximately 45%. This enhancement of Ig secretion was SP-specific, as evidenced by the ability of 1000-fold excess of SP antagonist to block SP-induced, but not LPS-induced, Ig production. Clearly, SP could act synergistically with LPS to enhance Ig secretion; therefore, we questioned whether this augmentation was also reflected at the level of H chain mRNA expression. 10(-9)M SP induced modest increases (50 to 60%) in mu-chain mRNA expression by LPS-stimulated 5F5 cells when compared with cells stimulated with LPS alone. The 4F10 cells did not display this magnitude of difference for alpha-chain mRNA expression. Thus, although SP-induced increases of mu-chain mRNA by 5F5 cells may contribute to the increased Ig secretion observed by these LPS-activated lymphocytes, it is unlikely that increased mRNA expression can totally account for the threefold increases in secretion that were observed.
Cyclic AMP directly inhibits IL-2 receptor expression in human T cells: expression of both p55 and p75 subunits is affected.
Authors: Krause D S , Deutsch C
Published: December 2022

Using purified human T lymphocytes stimulated in serum-free media with adhered anti-CD3 + exogenous IL-2, we have shown that elevated [cAMP]i (mimicked by CPT-cAMP or induced by the physiological agonist PGE2) directly inhibits mitogen-induced 1) [3H]thymidine incorporation by PBMC, purified T cells, and isolated CD4+ and CD8+ T cell subpopulations; 2) expression of both high- and low-affinity IL-2 receptors; 3) plasma membrane expression of both p55 and p75 subunits of the IL-2 receptor; and 4) expression of p55 mRNA, but not p75 mRNA. The decrease in p55 mRNA is not due to enhanced mRNA metabolism. We conclude that elevated [cAMP]i, acting directly on T cells, inhibits mitogenesis by decreasing IL-2 receptor expression. We discuss the possible physiological relevance for the multiple stages of T cell activation that are sensitive to elevated [cAMP]i.
The ability of cultured Langerhans cells to process and present protein antigens is MHC-dependent.
Authors: Aiba S , Katz S I
Published: December 2022

There is controversy regarding the ability of short term (2 to 3 days) cultured epidermal Langerhans cells (cLC) to process and present intact protein Ag to primed T cells. Some studies have shown that cLC are potent APC for both haptens and intact protein Ag, whereas in others cLC have been unable to process and present intact protein Ag. In an attempt to resolve this controversy, we tested the ability of Langerhans cells from several strains of mice to process and present intact protein Ag to T cell clones and T cell hybridomas. We found that both cLC and freshly prepared Langerhans cells from various Iak mice, including BALB.k mice, process and present intact protein antigens (i.e., hen egg lysozyme, cytochrome c, and OVA) to T cells. These functions are retained in cLC cultured for 7 days. In contrast, cLC from Iad mice do not process intact protein Ag, such as hen egg lysozyme and myoglobin, although they can present relevant peptides to specific T cells and are potent stimulators of allogeneic responses. Furthermore, cLC from (Iak x Iad)F1 mice process and present intact protein Ag to Iak-restricted T cells, but not to Iad-restricted T cells. Although cLC that processed and presented intact protein Ag to T cells exhibited enhanced class II MHC expression, they were, on a per cell basis, somewhat less efficient than were fresh Langerhans cells. Finally, we found that if Iad Langerhans cells are pulsed with intact protein Ag and then cultured for 3 days, they are then fully capable of inducing Ag- and MHC-specific T cell proliferation.
Regulation of expression of CD27, a T cell-specific member of a novel family of membrane receptors.
Authors: de Jong R , Loenen W A , Brouwer M , van Emmerik L , de Vries E F , Borst J , van Lier R A
Published: December 2022

CD27 belongs to a newly defined family of transmembrane R, including the nerve growth factor R, two distinct TNF R and CD40. The function of CD27 is unknown, but on the basis of structural and functional properties, we postulate that it plays a role in the events subsequent to T cell activation, possibly as a cytokine R. We have analyzed the mechanisms underlying the regulation of CD27 protein expression. Membrane expression of CD27 strongly increases after T cell activation via the TCR/CD3 complex or the CD2 molecule. In contrast, direct stimulation of protein kinase C by phorbol esters markedly down-regulates CD27 surface expression. This down-regulation most likely does not result from CD27 phosphorylation, because both anti-CD3 mAb and PMA induce hyperphosphorylation of CD27 on serine residues. Rather, membrane expression seems to be regulated primarily at the RNA level. Stimulation of T cells with anti-CD3 mAb strongly increases steady state CD27 mRNA levels, whereas PMA treatment greatly reduces these transcript levels. Dissection of the TCR/CD3-induced signaling pathways showed that cytoplasmic cAMP as well as Ca2+ concentrations contribute to the increase of CD27 expression. These data indicate that upon Ag-specific T cell stimulation, membrane expression of CD27 is regulated at the RNA level through the joint action of distinct TCR/CD3-associated signaling pathways.
Reconstitution of an active surface CD2 by DNA transfer in CD2-CD3+ Jurkat cells facilitates CD3-T cell receptor-mediated IL-2 production.
Authors: Makni H , Malter J S , Reed J C , Nobuhiko S , Lang G , Kioussis D , Trinchieri G , Kamoun M
Published: December 2022

To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti-CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL-2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR-induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex.
Aspirin-like drugs prime human T cells. Modulation of intracellular calcium concentrations.
Authors: Flescher E , Fossum D , Gray P J , Fernandes G , Harper M J , Talal N
Published: December 2022

Aspirin-like drugs (ALD) enhance T cell proliferation by suppressing PG production in monocytes. Normal human T cells do not produce any eicosanoids. Therefore we studied whether ALD would affect purified T cells directly. We found that ALD enhanced the proliferation and IL-2 production of T cells in the absence of monocytes. This effect did not depend on arachidonic acid metabolism as no lipoxygenase products and only nonsuppressive levels of cyclooxygenase products were detected in T cell cultures. Several possible mechanisms of the ALD effect were ruled out including 1) enhanced mitogen binding, 2) induction of activation markers (IL-2R, transferrin receptor, HLA-DR) on the cell surface, 3) down-regulation of suppressor cells. ALD caused a rise in [Ca2+]i which appeared to reflect an influx of Ca2+ from the extracellular milieu and was more pronounced in CD4+ cells. The rise in intracellular levels of Ca2+, that is considered a necessary second messenger for T cell activation, may prime these cells for an enhanced response to mitogens. In addition, ALD increased T cell membrane fluidity but only at higher concentrations than those found to enhance proliferation. The pharmacologic effect of ALD on T cells presents a possible new immunoenhancing potential of these drugs and may have therapeutic use in immunosuppressed individuals.
Effects of transforming growth factor-beta and IL-1 alpha on prostaglandin synthesis in serum-deprived osteoblastic cells.
Authors: Marusić A , Kalinowski J F , Harrison J R , Centrella M , Raisz L G , Lorenzo J A
Published: December 2022

We investigated the effects that the combination of IL-1 alpha and transforming growth factor-beta (TGF-beta) had on PGE2 production in a murine clonal osteoblastic cell line MC3T3-E1 and primary rat calvarial osteoblast-like cells. In serum-supplemented medium, IL-1 alpha was a potent stimulator of PGE2 production in MC3T3-E1 cells (50-fold increase with 0.1 ng/ml). TGF-beta (10 ng/ml) had only a small effect alone and no additional effect on IL-1 alpha-induced responses. In serum-deprived MC3T3-E1 cells, PGE2 responses to IL-1 alpha were either absent or markedly reduced. TGF-beta alone had small effects. However, simultaneous addition of TGF-beta with IL-1 alpha to MC3T3-E1 cells partially restored the ability of IL-1 alpha to generate a PGE2 response (10-fold increase in PGE2 with 0.1 ng/ml of both IL-1 alpha and TGF-beta). As with MC3T3-E1 cells, serum-deprived primary fetal rat calvarial osteoblastic cells also did not respond to IL-1 alpha, unless TGF-beta was present in the medium (sixfold increase in PGE2 with 0.1 ng/ml IL-1 alpha and 10 ng/ml TGF-beta). The synergistic effect of TGF-beta and IL-1 alpha was specific for PGE2 responses, because these factors did not synergistically affect cell proliferation, collagen and noncollagen protein synthesis, or alkaline phosphatase activity. The observed synergy was not associated with changes in the steady state cyclooxygenase (PGH synthase) mRNA levels. However, it did correlate with increased release of [3H]arachidonic acid from prelabeled serum-depleted MC3T3-E1 cells. Hence, the synergistic interactions of IL-1 alpha and TGF-beta on PGE2 appear to occur through an increase in the release of arachidonic acid substrate from phospholipid pools. These effects may be important for both normal bone turnover and the responses of bone to inflammatory and immune stimuli.
Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system.
Authors: Nishimura Y , Mayumi H , Tomita Y , Eto M , Maeda T , Nomoto K
Published: December 2022

The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR.
Epitopes of the p70 and p80 (Ku) lupus autoantigens.
Authors: Reeves W H , Pierani A , Chou C H , Ng T , Nicastri C , Roeder R G , Sthoeger Z M
Published: December 2022

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.
Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages.
Authors: Maródi L , Korchak H M , Johnston R B
Published: December 2022

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.
Probing VH gene-family utilization in human peripheral B cells by in situ hybridization.
Authors: Zouali M , Theze J
Published: December 2022

Recent studies on the genomic organization of the human Ig VH locus have revealed the presence of an important proportion of VH pseudogenes and a high degree of interspersion among VH gene-family members. The mechanisms of selection of VH genes expressed by differentiating B cells remain to be elucidated. We have made use of RNA-RNA in situ hybridization to probe the repertoire of VH gene-families assembled in peripheral B cells of normal adults. Cells were in vitro activated with mitogens and then hybridized to [35S]-labeled anti-sense RNA probes specific for C mu and C gamma genes, and for the six known human VH gene-families. We found that the numbers of cells expressing C mu and C gamma mRNA were similar to the total numbers of cells expressing members of the six VH gene-families. Therefore, the six known VH gene-families represent essentially the human VH locus. We also found that expression of VH gene-families does not closely correlate with their relative genomic complexity. This apparently biased expression may suggest that in some VH gene-families the ratio of pseudogenes/functional genes is particularly high, and/or that regulatory mechanisms play a major role in shaping the available VH gene repertoire in differentiating B cells.
Anti-C-reactive protein inhibits the calcium-dependent stage of natural killer cell activation.
Authors: Hamoudi W H , Baum L L
Published: December 2022

The studies described in this publication were designed to determine which stage of the NK lytic mechanism is inhibited by anti-C-reactive protein (CRP). Although anti-CRP prevents target cell lysis, it does not block E:T cell conjugate formation. In parallel experiments, the number of conjugates observed in the presence of anti-CRP was normal, whereas the number of target cells killed by this same group of effector cells was greatly inhibited. Since an early stage of NK-mediated lysis requires calcium, conjugates can be synchronized by incubating effector and target cells in the absence of calcium. When conjugates were formed in the absence of calcium, and anti-CRP and calcium were then added to cultures at the same time, anti-CRP inhibited maximally. Anti-CRP continued to inhibit somewhat throughout the calcium-dependent stage but did not block lysis when added after the completion of calcium requiring events. Events that are blocked by anti-CRP must be required for the generation of NK cytotoxic factor because anti-CRP blocks the production of this factor. Once generated, however, anti-CRP does not block the activity of NK cytotoxic factor. This evidence indicates that anti-CRP blocks NK-mediated lysis at the calcium dependent stage of lysis. Events that follow this stage in the lytic process are also inhibited.
IL-4 is required for the IgE and IgG1 increase and IgG1 autoantibody formation in mice treated with mercuric chloride.
Authors: Ochel M , Vohr H W , Pfeiffer C , Gleichmann E
Published: December 2022

Previous studies have established that in susceptible mouse strains, such as A.SW (H-2s), repeated injections of subtoxic doses of HgCl2 induce increased serum levels of total IgE and IgG1, high serum titers of antinuclear autoantibodies (ANo1A), and immune-complex glomerulonephritis. Moreover, it has been shown that susceptibility is determined by H-2As and that Th cells are required for the induction of these immunopathologic alterations by HgCl2. In the present study we showed that treatment in vivo with anti-IL-4 mAb completely abrogated the HgCl2-induced increase in total IgE and partially inhibited the increase in IgG1, but failed to suppress the increase in IgG2A. Furthermore, we showed that IL-4 influences the pattern of IgG subclass distribution among ANo1A of HgCl2-treated mice. Whereas treatment with anti-IL-4 mAb significantly reduced the titers of IgG1 ANolA, it increased those of IgG2A, IgG2B, and IgG3 ANolA. Thus, these results show that IL-4 contributes to the optimal formation in vivo of murine IgG1 and that it is involved in the autoantibody formation of a systemic autoimmune disease. The available evidence suggests that HgCl2 induces an increased production of IL-4 by Th2 cells. If this is correct, it implies that MHC class II alleles determine whether the preferential response to HgCl2 is made by Th1 or Th2 cells and, hence, the type of immunopathologic alterations ensuing.
Studies on the induction of anterior chamber-associated immune deviation (ACAID). II. Eye-derived cells participate in generating blood-borne signals that induce ACAID.
Authors: Wilbanks G A , Mammolenti M , Streilein J W
Published: December 2022

Despite an ever increasing body of information concerning general in vivo immune reactivity to exogenous Ag, relatively little is known concerning regional tissue influences on these responses. One expression of regionally distinct immune regulation is the deviant form of systemic immunity induced after the introduction of Ag, such as BSA, into the anterior chamber of the eye--anterior chamber-associated immune deviation (ACAID). ACAID is characterized by a selective, systemic immune deficiency in which both Ag-specific delayed hypersensitivity (DH) and complement-fixing antibody production are impaired by populations of Ts lymphocytes, although other immune effector modalities are preserved. Recent evidence demonstrates that the blood of mice that receive an anterior chamber injection of BSA 48 h earlier contains leukocytes capable of inducing cell-mediated Ag-specific efferent suppression of DH similar to that seen in mice with ACAID. Additional analysis of these bloodborne suppression-inducing leukocytes revealed them to express the mature macrophage/monocyte marker F4/80. On the basis of these findings, we wished to determine the source of the ACAID-inducing cells present in the peripheral blood of mice after anterior chamber inoculation of BSA. The data reveal that cells harvested from the normal eye are capable of selective induction of DH suppressive activity similar to that seen in ACAID. Interestingly, this property is unique to F4/80 expressing cells of the iris and ciliary body (I/CB). F4/80- I/CB cells do not possess this property. Although the F4/80 expressing cells that are present in extraocular sites do not constitutively possess ACAID-inducing properties, exposure of these cells to the anterior chamber microenvironment endows them with this ability. Finally, I/CB cells exposed to Ag in situ, via direct anterior chamber Ag inoculation, induce suppressed Ag-specific DH reactivity when infused into naive syngeneic recipients--a form of systemic immune deviation similar to that seen in mice with ACAID. The results presented here represent the first in vivo description of APC, altered by factors in a local microenvironment, that can initiate a systemic immune response characterized by Ag-specific immune suppression.
Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines.
Authors: Mackiewicz A , Speroff T , Ganapathi M K , Kushner I
Published: December 2022

We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.
Involvement of the 55- and 75-kDa tumor necrosis factor receptors in the generation of lymphokine-activated killer cell activity and proliferation of natural killer cells.
Authors: Naume B , Shalaby R , Lesslauer W , Espevik T
Published: December 2022

In this study we investigated the expression of the 55 kDa (p55) and the 75 kDa (p75) TNF receptors in CD56+ NK cells and their role in NK and lymphokine-activated killer cells cell functions. By using mAb against the p55 and p75 TNF-R, NK cells were found to express both p55 and p75 upon activation, and both receptors were involved in the generation of lymphokine-activated killer cells activity. Proliferative activity of IL-2 stimulated NK cells was inhibited by anti-TNF-alpha mAb, indicating that endogenously produced TNF-alpha is important for optimal proliferation of NK cells. Furthermore, addition of rTNF-alpha increased the IL-2-induced proliferation of NK cells. mAb to p55 and p75 inhibited the IL-2-induced proliferation indicating that both TNF-R are involved in mediating this effect.
Serologic and chemical differentiation of human lambda III light chain variable regions.
Authors: Eulitz M , Murphy C , Weiss D T , Solomon A
Published: December 2022

Human lambda L chains of a major V lambda subgroup, V lambda III, have been differentiated serologically and chemically into three V lambda III sub-subgroups designated V lambda IIIa, V lambda IIIb, and V lambda IIIc. Antisera prepared against lambda III Bence Jones proteins were obtained that recognized distinctive V lambda III-related epitopes expressed by monoclonal lambda III L chains. After appropriate absorption, these reagents were rendered specific for three distinct populations of lambda III proteins--lambda IIIa, lambda IIIb, and lambda IIIc. The antisera were used in comparative immunodiffusion analyses of 28 monoclonal lambda III L chains, 10 of which were classified as lambda IIIa, 4 as lambda IIIb, and 14 as lambda IIIc. The isotypic nature of the three lambda III sub-subgroups was demonstrated serologically through analyses of lambda-chains derived from the serum IgG molecules of normal individuals. The amino acid sequences of five serologically classified lambda III chains, which included members of the three V lambda III sub-subgroups, had been previously determined. This information, in addition to our establishment of the complete (or virtually complete) V region sequence of 15 and the partial sequence of eight other lambda IIIa, lambda IIIb, and lambda IIIc proteins, made it possible to correlate chemical data with serologic classification. Proteins within each of the three serologically-classified lambda III sub-subgroups typically possessed a high degree (approximately 83%) of intra-sub-subgroup sequence homology that included both framework and complementarity determining region residues. Furthermore, within the framework and complementarity determining regions, sub-subgroup-specific residues were identified. Taken together, these data reveal that the human V lambda III genome consists of (at least) three distinct V lambda IIIa, V lambda IIIb, and V lambda IIIc germline genes that encode for lambda IIIa, lambda IIIb, and lambda IIIc L chains, respectively.
Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.
Authors: Walker B A , Hagenlocker B E , Ward P A
Published: December 2022

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.
Cyclosporin A modulates the course of woodchuck hepatitis virus infection and induces chronicity.
Authors: Cote P J , Korba B E , Steinberg H , Ramirez-Mejia C , Baldwin B , Hornbuckle W E , Tennant B C , Gerin J L
Published: December 2022

Immunosuppression is known to influence the state of chronic hepatitis B virus infection, and is thought to increase the risk of developing chronic infection in newly exposed individuals. Cyclosporin A (CsA), an immunosuppressive agent that inhibits Th cell function, was administered to woodchucks chronically infected with woodchuck hepatitis virus (WHV), and resulted in a decreased severity of chronic hepatitis and an increased viremia during the treatment. Adult woodchucks inoculated with WHV and given CsA for 14 wk had increased viremias, decreased acute phase liver injury, and developed chronic infections at a higher rate compared with immunocompetent woodchucks given virus alone (chronicity in seven of seven WHV + CsA + vs zero of nine WHV + CsA-; p less than 0.001). These results in a relevant animal model of hepatitis B virus infection indicate: 1) that liver injury in acute hepadnavirus infections is immune-mediated and not a direct cytopathic effect of virus replication; 2) that Th cells function in the inflammatory response and in the immunologic control of hepadnavirus infection; and 3) that suppression of Th cell function in acute hepadnavirus infection decreases liver injury but alters the outcome of infection in favor of chronicity. These results also suggest continued challenges in the application of CsA in liver transplantation for hepatitis B virus-induced diseases.
Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.
Authors: Lancki D W , Hsieh C S , Fitch F W
Published: December 2022

Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.
Interaction of CD2 with its ligand lymphocyte function-associated antigen-3 induces adenosine 3',5'-cyclic monophosphate production in T lymphocytes.
Authors: Hahn W C , Rosenstein Y , Burakoff S J , Bierer B E
Published: December 2022

CD2 (T11, the T cell E receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, is a T cell-specific surface protein that plays an important role in T lymphocyte adhesion, signal transduction, and differentiation. A natural ligand of CD2 is lymphocyte function associated Ag-3 (LFA-3 (CD58)), a widely expressed glycoprotein of 50 to 70 kDa. The physiologic interaction of CD2 with LFA-3 functions to increase intercellular adhesion and plays a role in T cell activation. This interaction, however, in the absence of other stimuli, has not previously been shown to induce intracellular signals such as Ca2+ mobilization or IL-2 production. To investigate whether cAMP may play a role in ligand-triggered CD2-mediated signal transduction, we have studied the ability of purified LFA-3 and anti-CD2 mAb to induce changes in intracellular cAMP content in murine Ag-specific T cell hybridomas that stably express wild-type and mutated human CD2 molecules. By using a RIA sensitive to the femtomolar range and specific for cAMP, we demonstrate that purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3 alone to CD2-expressing hybridoma cells, however, did not stimulate phosphatidylinositol turnover nor IL-2 production. The cytoplasmic domain of CD2 is necessary for these ligand-induced cAMP changes, demonstrating that LFA-3 binding to CD2 transduces a signal to the cell. Experiments using the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine showed that CD2-mediated regulation of cAMP levels occurs primarily by the stimulation of cAMP production rather than by the inhibition of cAMP degradation. These results demonstrate that the interaction of LFA-3 with CD2, in the absence of other stimuli, is capable of initiating intracellular biochemical changes and suggest that CD2/LFA-3 interactions may regulate T cell function at least in part through the generation of intracellular cAMP.
A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain.
Authors: Choo S Y , Fan L A , Hansen J A
Published: December 2022

There are six known HLA-B alleles that share the HLA-B27 allospecificity, yet differ by one to six amino acid substitutions. Each of these B27 alleles can be readily assigned by one of the six representative IEF patterns. Two unrelated individuals, LH and HS, express B27 Ag that appear to be identical by IEF, but an HLA-B27 alloreactive CTL clone I-73 was found to react differently with these cells, suggesting these B27 molecules are not identical. We sequenced polymerase chain reaction-amplified B27 cDNA clones obtained from HS and compared its deduced amino acid sequence (B27-HS) with the B27 sequence of LH (B27-LH) which was previously designated the B*2701 allele. B27-HS and B27-LH differ by eight amino acids; three in alpha 1 domain and five in alpha 2 domain. These amino acid substitutions of B27-HS altered T cell recognition but not the B27 serologic epitope or IEF pattern. B27-HS differs from the six known B27 alleles by five to eight amino acid substitutions, and thus it represents the seventh allele of the HLA-B27 Ag family. This novel B27 allele might have been derived from a gene conversion event. Previously, two amino acid residues at positions 70 and 97 were suggested to be specific for B27 Ag family. B27-HS now reveals that Lys at position 70 is specific for B27 but Asn at position 97 is not. We propose that the region around position 70 might be crucial in determining the B27 serologic epitope and possibly in peptide Ag binding. This study also demonstrates that class I molecules of the same Ag specificity sharing an indistinguishable IEF pattern are not necessarily identical, and indicates that only the definitive determination of primary structure would identify all the class I alleles that are functionally relevant in regard to alloreactivity, T cell restriction, and disease association.
Conformational and structural characteristics of peptides binding to HLA-DR molecules.
Authors: Hill C M , Hayball J D , Allison A A , Rothbard J B
Published: December 2022

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.
Comparison of structural requirements for interaction of the same peptide with I-Ek and I-Ed molecules in the activation of MHC class II-restricted T cells.
Authors: Leighton J , Sette A , Sidney J , Appella E , Ehrhardt C , Fuchs S , Adorini L
Published: December 2022

We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107-116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules.
Phagocytosis and stimulation of the respiratory burst by phorbol diester initiate S-thiolation of specific proteins in macrophages.
Authors: Rokutan K , Thomas J A , Johnston R B
Published: December 2022

Addition of chemical oxidants to cells in culture has been shown to induce binding of low-molecular-weight thiols to reactive sulfhydryls on proteins in a process termed S-thiolation. We found that stimulation of the respiratory burst in mouse macrophages, with release of O2-, resulted in S-thiolation of several proteins, most prominently three with molecular weights of 74, 33, and 22 kDa. One protein (28 kDa) was S-thiolated without addition of an exogenous stimulus. Exposure of cells to concentrations of hydrogen peroxide like those released in the respiratory burst induced S-thiolation of these same proteins. S-thiolation and release of O2- began at approximately the same time. Stimulation of LPS-elicited macrophages induced prominent S-thiolation of three different proteins (38, 30, and 21 kDa). Under the conditions of these experiments, there was no detectable increase in glutathione disulfide and a negligible decrease in glutathione, which suggests that S-thiolation can occur without significant perturbation of the glutathione peroxidase/reductase cycle. S-thiolation of proteins could help protect the macrophage against the autoxidative damage associated with the respiratory burst. Modification of specific proteins by S-thiolation might serve to modulate cellular metabolic events.
Equilibrium binding of cytotoxic T lymphocytes to class I antigen.
Authors: Mescher M F , O'Rourke A M , Champoux P , Kane K P
Published: December 2022

Cloned cytotoxic T lymphocytes specifically bind to purified alloantigen that has been immobilized on a surface. When the time course was examined, it was found that binding reached a plateau level within about 1 h at 37 degrees C, at which time about 30% of the CTL were tightly adhered to the surface. Analysis of the properties of binding demonstrated that this does not simply result because only a fraction of the cells in the clonal population are capable of binding. Instead, the binding is shown to result from an equilibrium involving tightly bound and unbound (or weakly bound) cells. Thus, the cells cycle between a tightly bound and unbound state, despite continuous contact with the Ag-bearing surface. The results suggest that dissociation of the bound cells may be an actively signaled event. A model that could account for these results based on activated CD8 binding is discussed.