bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of histamine H1 binding sites on human T lymphocytes by means of 125I-iodobolpyramine. Preferential expression of H1 receptors on CD8 T lymphocytes.
Authors: Villemain F M , Bach J F , Chatenoud L M
Published: December 2022

The presence of histamine H1 receptors on lymphocytes has been indirectly suggested by the various effects of agonists or antagonists on the functionally distinct T lymphocyte subsets. Recently, a new H1 antagonist, 125I-iodobolpyramine, whose structure is similar to mepyramine, has become available for the detection of H1 receptors in guinea pig brain. When using 125I-iodobolpyramine on human T lymphocytes, the presence of a single highly specific H1 binding site was evidenced. The binding of 125I-iodobolpyramine to human T cells was reversible when using 1000-fold excess of the cold H1 antagonist, d-chlorpheniramine. Binding saturation was achieved at 0.60-0.65 nM of 125I-iodobolpyramine, the binding equilibrium was reached in 20-30 min at 27 degrees C. The dissociation constant was KD = 0.41 +/- 0.07 (mean +/- SE) and the number of receptors per T cell was 3407 +/- 592 (mean +/- SE) as deduced from saturation and kinetic curves. In competition experiments using a panel of H1 ligands, the T cell binding sites detected by 125I-iodobolpyramine showed a pharmacological behavior characteristic of histamine H1 receptors. It was of particular interest that 125I-iodobolpyramine binding displayed clearcut stereoselectivity as assessed by the higher affinity of the d-configuration of chlorpheniramine than the l form. Study of purified CD4 and CD8 T cells showed that twice as much H1 histamine receptors were expressed by CD8 T lymphocytes (6615 +/- 1125) as compared to CD4 T cells (3545 +/- 459). These results underline the need for studying the functional properties of such pharmacologically defined T lymphocyte H1 binding sites.
Signal transduction via leukocyte antigen CD43 (sialophorin). Feedback regulation by protein kinase C.
Authors: Wong R C , Remold-O'Donnell E , Vercelli D , Sancho J , Terhorst C , Rosen F , Geha R , Chatila T
Published: December 2022

CD43 is a constitutively phosphorylated 115-kDa sialoglycoprotein expressed on a variety of blood cells including lymphocytes and monocytes. L10, a mAb directed against CD43, triggers T cell activation and enhances hydrogen peroxide production in monocytes. Activation of mononuclear cells by L10 initiates phosphoinositides hydrolysis, C2+ mobilization, and protein kinase C (PKC) activation. In turn, activated PKC hyperphosphorylates CD43, suggesting a potential role for PKC in the regulation of signaling via CD43. To address this issue, we have analyzed the effect of PKC activation by the tumor promoter PMA on L10-triggered rise in intracellular free Ca2+ concentrations ([Ca2+]i). Treatment of mononuclear cells with PMA profoundly inhibited the increase in [Ca2+]i induced by L10. The inhibition of CD43-mediated signaling by PMA was due, in part, to uncoupling of CD43 from the signal-transducing G protein. This was evidenced by the comparatively modest inhibition by PMA of the increase in [Ca2+]i induced by the direct G protein activator AlF4-. PMA treatment did not affect the surface expression of CD43. However, it induced the hyperphosphorylation of CD43, the extent of which correlated with the inhibition of CD43-mediated increase in [Ca2+]i. Staurosporine, a potent inhibitor of PKC, abrogated the hyperphosphorylation of CD43 and normalized CD43-mediated signaling in PMA-treated cells. Significantly, in the absence of PMA, staurosporine enhanced the rise in [Ca2+]i triggered by L10, suggesting that engagement of CD43 by activating ligands results in feedback inhibition by PKC. It is concluded that activation of PKC inhibits signaling via CD43 by mechanisms involving phosphorylation and uncoupling of CD43 from the signal-transducing apparatus and by distal, post-receptor events.
Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.
Authors: Oftung F , Shinnick T M , Mustafa A S , Lundin K E , Godal T , Nerland A H
Published: December 2022

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.
Induction of macrophage Ia expression in vivo by a synthetic block copolymer, L81.
Authors: Howerton D A , Hunter R L , Ziegler H K , Check I J
Published: December 2022

Certain synthetic, nonionic, block copolymers of polyoxypropylene and polyoxyethylene are potent immunoadjuvants. While investigating their mechanisms of action, we found that one of these copolymers, L81, induced the expression of macrophage Ia in vivo. L81 is a 2750-Da, linear copolymer with a 43-unit core of hydrophobic polyoxypropylene flanked on each side by 3 U of hydrophilic polyoxyethylene. It induced threefold to sevenfold increases in the proportion of peritoneal macrophages expressing I-A from 5 to 7 days after an i.p. injection of 5 mg. As little as 1 mg caused a twofold increase. I-A density increased with time after injection of L81. Macrophages induced by L81 actively synthesized I-A, showing an 18-fold increase in biosynthetic capacity. Ia induction did not require the presence of mature T lymphocytes, because similar increases in I-A expression were seen in athymic and euthymic mice. L81-induced macrophages were 10-fold more effective than normal macrophages in presenting Ag to a T cell hybridoma. Other functional studies showed that they were primed for the secretion of superoxide ion and could be stimulated in vitro by IFN-gamma and LPS to lyse tumor target cells. These results suggest that the induction of macrophage Ia expression by L81 may play a role in its activity as an immunoadjuvant.
Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.
Authors: June C H , Fletcher M C , Ledbetter J A , Samelson L E
Published: December 2022

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.
Alloreactive murine CD8+ T cell clones secrete the Th1 pattern of cytokines.
Authors: Fong T A , Mosmann T R
Published: December 2022

A large panel of CD8+ mouse T cell clones expressed the cytokine synthesis pattern characteristic of Th1 clones. CD8+ clones synthesized IFN-gamma and lymphotoxin at levels similar to Th1 clones, whereas IL-2 was synthesized by only 50% of the clones and at significantly lower levels compared to Th1 clones. CD8+ clones also produced substantial amounts of granulocyte/macrophage-CSF, TY5, P500, and TNF-alpha which are expressed preferentially by Th1 clones and at lower levels by Th2 clones. The level of IL-3 produced by CD8+ clones was approximately 10% of that produced by Th1 and Th2 clones. Some CD8+ clones expressed low levels of the Th2-preferential product preproenkelphalin. None of the CD8+ clones expressed detectable levels of the Th2-specific products IL-4, IL-5, and P600, and the great majority did not express IL-6. The cytokine profile of CD8+ clones is representative of that secreted by activated normal CD8+ splenocytes, which includes IFN-gamma, low levels of IL-2 and IL-3 but no IL-4 or IL-5. Inasmuch as many Th1/Th2 functions are cytokine mediated, the striking similarity of the Th1 and CD8+ cytokine secretion patterns helps to explain why these two cell types share certain functions such as DTH, and also suggests that further common functions may be discovered in the future.
Tumor necrosis factor-alpha and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2.
Authors: Suda T , Murray R , Fischer M , Yokota T , Zlotnik A
Published: December 2022

Cytokines are known to play a key role in the development of several hemopoietic lineages including lymphocytes. Two cytokines: IL-4 (in the presence of PMA) and IL-7 have been shown to induce immature fetal thymocyte proliferation. It has also been suggested that IL-2 plays an important role in fetal T cell development. In this report, we investigated the effects of several cytokines (known to be growth factors for T-lineage cells) on fetal thymocyte proliferation. Our results indicate that: 1) TNF-alpha and a newly described cytokine, P40, enhance fetal thymocyte proliferation stimulated by IL-2 (but not IL-4 or IL-7). 2) The enhancement induced by P40 is not mediated by TNF-alpha because blocking antibodies against this cytokine failed to inhibit this response. 3) IL-4 inhibits fetal thymocyte proliferation in response to TNF-alpha + IL-2 or to IL-7 but not to P40 + IL-2. Finally, 4) the proliferating cells to all cytokine combinations used were Thy-1+. These observations suggest that these cytokine combinations induce independent pathways of T cell proliferation in the developing thymus.
The monoclonal antibody 41H16 detects the Leu 4 responder form of human Fc gamma RII.
Authors: Gosselin E J , Brown M F , Anderson C L , Zipf T F , Guyre P M
Published: December 2022

Human FcR for IgG can be divided into three classes (Fc gamma RI, II, and III) based on their structure and reactivity with mAb. Fc gamma RII can be further subdivided into two categories based on functional and biochemical assays. These two Fc gamma RII subtypes were initially recognized by the failure of T cells from 40% of individuals to proliferate in response to mAb Leu 4 (mouse IgG1, anti-CD3), a response that requires the binding of the Fc region of the Leu 4 mAb to Fc gamma RII on monocyte accessory cells. Inas-much as mouse IgG1, does not bind efficiently to the nonresponder form of Fc gamma RII, mAb Leu 4 is unable to induce proliferation in these individuals. IEF data on Fc gamma RII from Leu 4 responder and nonresponder individuals suggested that the structural gene for Fc gamma RII consisted of two allelic forms R (responder) and N (nonresponder) producing the phenotypes RR, RN, and NN. Thus, exclusive expression of the nonresponder allele in monocytes of "nonresponder" individuals, appeared to be responsible for the lack of proliferation observed. In cooperation with the IVth International Conference on Human Leukocyte Differentiation Antigens, we analyzed CDw32 mAb to determine if they could distinguish the responder and nonresponder forms of Fc gamma RII. We report that mAb 41H16 binds preferentially to the responder allotypic form of Fc gamma RII expressed on human monocytes. When quantitative flow cytometry is used to measure the binding of both mAb 41H16 (responder Fc gamma RII) and mAb IV.3 (all myeloid cell Fc gamma RII), we are able to subdivide the responder population into homozygous and heterozygous responders. In addition, mAb 41H16 blocks the binding of mAb IV.3 to monocytes and inhibits proliferation when added to cells before addition of mAb Leu 4. We also show that polymorphonuclear leukocytes and platelets have the same allotypic differences in the binding of 41H16 as do monocytes. However, a subset of lymphocytes (previously shown to be B cells) expresses the 41H16 epitope with no evidence for donor to donor variability.
Influence of microvascular adherence on neutrophil leukotriene generation. Evidence for cooperative eicosanoid synthesis.
Authors: Grimminger F , Kreusler B , Schneider U , Becker G , Seeger W
Published: December 2022

Profile and quantity of leukotriene (LT) and hydroxyeicosatetraenoic acid (HETE) generation upon selective stimulation of isolated polymorphonuclear neutrophils (PMN) compared with neutrophils in a model of pulmonary leukostasis were investigated. Freshly prepared human PMN (2 x 10(8) were injected into the pulmonary artery of isolated, ventilated, and bloodfree perfused rabbit lungs, resulting in nearly quantitative sticking in the microvasculature. The sequestered neutrophils and, in parallel, aliquots of isolated PMN were stimulated with mAb in the presence of C, known to activate PMN arachidonate metabolism via formation of membrane attack complexes. In the isolated cells, a typical LT profile including LTB4 and its omega-oxidation products, 5-HETE and nonenzymatic hydrolysis products of LTA4 was evoked. The latter indicate secretion of LTA4 in considerable amounts. In the model of pulmonary leukostasis, no nonenzymatic LTA4-derivatives were detected, coincident with a predominance of cysteinyl-LT. This finding gives indirect evidence for an efficient LTA4-transfer between PMN feeder cells and vascular acceptor cells with glutathione-S-transferase activity. Moreover, a threefold increase in the total amount of LTA4-derived products was noted in the model of leukostasis, paralleled by a marked decrease in 5-HETE liberation. This effect was further enhanced by inhibition of lung cyclooxygenase. These findings were corroborated in a homologous system, in which rabbit PMN, sticking in the rabbit lung microvasculature, were stimulated with calcium-ionophore A23187. Collectively, these data suggest a complex interaction between microvascular tissue and adhering neutrophils in LT synthesis, involving transcellular LTA4-shift, modulation of the PMN 5-lipoxygenase pathway, and amplification of LT generation. These findings may be relevant for inflammatory events with neutrophils involved.
The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2.
Authors: Gross T J , Sitrin R G
Published: December 2022

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.
Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides.
Authors: Mullmann T J , Siegel M I , Egan R W , Billah M M
Published: December 2022

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.
Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination.
Authors: Suter M , Becker R S , Knight K L
Published: December 2022

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.
The expression of a tissue-specific self-peptide is required for allo-recognition.
Authors: Molina I J , Huber B T
Published: December 2022

We have compared the functional properties of I-Ad expressed on different cell types. Specifically, we have transfected I-A alpha d and I-A beta d cDNA into a panel of T cell thymomas of various phenotypes. Excellent class II surface expression was achieved in all T cell tumors, equivalent in level to that found on the B cell lymphoma A20. Interestingly, however, two allo-I-Ad-specific Thy differed in their recognition of the transfected tumor cells: whereas the 42H11 T cell hybridoma (THy) was stimulated very efficiently by all transfectants, the RK38.2.2 Thy did not react to any of them. Both THy responded equally well to I-Ad on A20 B lymphoma cells. Purified macrophages isolated from various sources were also differentially recognized by the two THy, although there was only a quantitative difference in stimulation. Taken together, these results are best interpreted to show that the TCR of the RK38.2.2 THy is specific for I-Ad in the context of a B cell-specific determinant, possibly a self-peptide that is naturally associated with Ia. A cross-reactive molecule could be expressed by macrophages and COS-1 cells, but not by T cells.
Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide.
Authors: Chodakewitz J A , Lacy J , Edwards S E , Birchall N , Coleman D L
Published: December 2022

CSF have a broad range of effects on differentiated cells outside the bone marrow. Site-specific elaboration of these factors may influence local immune reactions. Keratinocytes have been demonstrated to produce a number of immunoactive cytokines, including factors capable of modifying macrophage function. We have previously identified at least two products of keratinocytes that induce DNA synthesis by elicited peritoneal macrophages; one factor has been identified as granulocyte-macrophage CSF. In the present study, the second keratinocyte product has been characterized and identified as macrophage-CSF (M-CSF). Conditioned media from cultures of normal human keratinocytes and the transformed murine keratinocyte cell line PAM 212 induce formation of macrophage colonies in soft agar as well as dose-dependent proliferation of the M-CSF-dependent cell line BAC1.2F5. The bioactivity in both assays is blocked by neutralizing anti-M-CSF antibody. Western blot analysis of cell lysates from both PAM 212 and normal human keratinocytes demonstrates multiple molecular mass forms of M-CSF (45 to 98 kDa). Northern blot analysis (PAM 212 cells) and in situ hybridization (normal keratinocytes) demonstrate expression of M-CSF mRNA. Stimulation of keratinocytes with LPS increases M-CSF synthesis as measured both by bioactivity and level of mRNA expression. Thus, both murine and human keratinocytes produce M-CSF in vitro. Furthermore, production of keratinocyte-derived M-CSF is increased by bacterial LPS. CSF production by keratinocytes may play an important role in regulating the cutaneous immune response.
Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules.
Authors: Codias E K , Malek T R
Published: December 2022

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.
IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells.
Authors: Martinez O M , Gibbons R S , Garovoy M R , Aronson F R
Published: December 2022

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.
IL-1 induces IL-1. IV. IFN-gamma suppresses IL-1 but not lipopolysaccharide-induced transcription of IL-1.
Authors: Schindler R , Ghezzi P , Dinarello C A
Published: December 2022

IL-1 induces its own gene expression in human PBMC, in cultured smooth muscle and in endothelial cells. IL-1-induced IL-1 may be part of a self-amplification or an autocrine growth factor in a variety of responses, and thus the endogenous regulation of IL-1 production likely contributes to the outcome of immunologic or inflammatory responses. In the present study, IFN-gamma consistently increased LPS-induced IL-1, but reduced the total amount of IL-1-induced IL-1 from PBMC. This reduction was observed in populations of adherent cells and cells selected with anti-Leu M3 antibody. On a molar basis, IFN-gamma and IFN-alpha 2 were equally effective in reducing IL-1-induced IL-1 synthesis. In PBMC of 24 human subjects, IFN-gamma also reduced PMA-induced IL-1 synthesis (67% decrease, p less than 0.001). The augmentation of LPS-stimulated IL-1 by IFN-gamma was observed only when added at the same time as LPS, but IFN-gamma could be added 6 h after stimulation with IL-1 and still suppress IL-1 production. LPS-induced mRNA for IL-1 beta was modestly enhanced by IFN-gamma whereas mRNA levels for TNF were markedly increased. In contrast, IFN-gamma suppressed mRNA accumulation for IL-beta after stimulation with IL-1 alpha by 60 to 95%. The addition of IFN-gamma 30 min before stimulation of PBMC with IL-1 suppressed IL-1 beta mRNA for up to 30 h. The half-life of IL-1 beta mRNA of approximately 2 h was not influenced by IFN-gamma. Thus, IFN-gamma reduces IL-1-induced IL-1 synthesis by suppressing IL-1-induced transcription, whereas the same concentrations of IFN-gamma augment LPS-induced IL-1 production by increasing transcription.
MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive xenoantisera.
Authors: Kaufman J , Skjoedt K , Salomonsen J , Simonsen M , Du Pasquier L , Parisot R , Riegert P
Published: December 2022

Rabbit antisera raised to human and chicken MHC molecules were used to immunoprecipitate cross-reactive molecules from biosynthetically and cell surface-labeled spleen and/or blood cells of representative vertebrate species. Five major points emerged: 1) There were many nonspecific cross-reactions using these techniques, so various criteria were developed to distinguish these from true MHC-like molecules. 2) Only very small subpopulations of immunogen-specific antibodies cross-reacted with MHC-like molecules in other nonmammalian species. These subpopulations were different for each species and even within a species, sometimes being so limited as to behave like alloantisera. This led to a very scattered pattern of true cross-reactions that sometimes failed to reflect the properties of the bulk antibody population. 3) Antisera containing antibodies to class II beta- and class I alpha-chains cross-reacted better and more widely than those to B-G, class II alpha and, in general, beta 2-microglobulin. 4) Some cross-reactive antibodies were clearly directed to epitopes on the surface of the mature heterodimers, but many seemed to recognize nonlinear cryptic determinants, presumably in the contact regions between the chains. These latter antibodies recognized biosynthetic intermediates and also a variety of unusual cell surface MHC-like molecules present in reptile and amphibian, but absent in the mammal and chicken cells tested. These included E homodimers whose relationship to chicken B-G molecules is unknown. 5) MHC-like molecules were identified in a bird, three reptiles, and two amphibians, but no molecules with the expected properties were found with these reagents in any of the fish tested.
MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive monoclonal antibodies.
Authors: Kaufman J , Ferrone S , Flajnik M , Kilb M , Völk H , Parisot R
Published: December 2022

mAb to human and mouse MHC molecules were tested for binding to blood or spleen cells of various nonmammalian vertebrates by immunofluorescence and flow cytometry. Those that bound were used to immunoprecipitate cross-reactive molecules from biosynthetically or cell surface-labeled spleen or blood cells. In addition, mAb to human MHC molecules were screened by Western blots. As expected from the results with xenoantisera, there were few mAb that cross-reacted, and many of these cross-reactions were not specific for MHC-like molecules. Less than 10% of the mAb tested bound to the cells of any particular species, with very few positive for more than one species. Of those mAb that bound cells, many failed to precipitate any radioactive bands, and most bands precipitated were not recognizable as MHC-like molecules. Five mAb reacted with Xenopus class II, one of which also immunoprecipitated axolotl class II. Another of these reacted with a candidate for class II in the lamprey, but this molecule had features unlike those expected for mammalian class II molecules. Four other mAb reacted with candidate molecules. in the trout and shark. None of the mouse alloantibodies immunoprecipitated nonmammalian vertebrate MHC-like molecules. In contrast to the results with most xenoantisera, the mAb cross-reacting with amphibian class II molecules recognized a number of different linear epitopes on the surface of the polymorphic non-Ig beta 1 domain of class II molecules. Few mAb recognized bands in Western blots of nonmammalian vertebrate cells and the candidate molecules from fish had features different from known mammalian MHC molecules.
Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase.
Authors: Schwartz L B , Bradford T R , Lee D C , Chlebowski J F
Published: December 2022

The catalytic activity of human tryptase, a mast cell neutral endoprotease, is expressed when the enzyme is in its tetrameric form, but is lost under physiologic conditions concomitant with a quaternary structural alteration involving conversion to a monomeric form. The associated changes in the CD spectra noted in the current study indicate accompanying alterations in the secondary structure of the protein. In particular, the progressive disappearance of the negative minimum centered at 228 nm suggests an effect on beta-sheet structure, which may be important for monomer-monomer interaction and/or stabilization of catalytic activity. Dextran sulfate, like heparin, stabilizes the catalytic activity and quaternary structure of tryptase and also maintains the native secondary structure of the enzyme at and beyond a temperature of 40 degrees C. Dextran sulfate-stabilized tryptase therefore was used as an immunogen to which were produced three murine mAb (B2, C11, and G4) recognizing the catalytically active form of the enzyme. Inactive tryptase bound to plastic microtiter wells was not recognized by any of the newly made antibodies, whereas inactive tryptase in solution was recognized by G4, which when biotinylated, could be used as a detector antibody in a sandwich ELISA for tryptase. Each of the newly made mAb recognized the catalytically active form of tryptase. Thus, alterations in epitopes, perhaps reflecting tertiary structural alterations as well as changes in secondary and quaternary conformations, occur with tryptase inactivation. A pragmatic result of these newly generated antibodies is the affinity purification to homogeneity of active tryptase by sequential chromatography with B2 coupled to CH-Sepharose and heparin-agarose. Tryptase purified by this technique had a specific activity with p-tosyl-L-arginine methyl ester of 117 +/- 9 U/mg and had 3.9 +/- 0.3 active sites per molecule of active enzyme (134,000 m.w.) as titrated with p-nitrophenyl-p'-guanidinobenzoate. The spectral and immunologic data in the current study are consistent with concerted conformational alterations in the secondary and tertiary as well as quaternary structures of tryptase associated with loss of catalytic activity. Failure to reverse any of these alterations with dextran sulfate suggests that the pathway of tetramer assembly in vivo is more complicated than simple subunit association.
Activation of the complement system during immunotherapy with recombinant IL-2. Relation to the development of side effects.
Authors: Thijs L G , Hack C E , Strack van Schijndel R J , Nuijens J H , Wolbink G J , Eerenberg-Belmer A J , Van der Vall H , Wagstaff J
Published: December 2022

Therapy with high doses of rIL-2 is complicated by the occurrence of hypotensive reactions and the development of a vascular leakage syndrome (VLS). In four patients, who together received seven cycles of high doses of IL-2 (up to 12 x 10(6) U per m2 per day), and who developed these side effects, we observed an unexpected increase in plasma levels of C3a, indicating activation of the complement system. C3a levels markedly increased during IL-2 therapy from 4 nmol/liter (mean level) before therapy to 23 nmol/liter at the end of the cycle. Activation of C3 occurred via the classical pathway inasmuch as C4a levels also increased during therapy. Mean daily C3a levels correlated with signs of the VLS, such as daily weight gain (p less than 0.001) and albumin levels (inverse correlation, p less than 0.001). In five additional patients, who together received seven cycles of lower doses of IL-2 (2 x 10(6) U per m2 per day) and who did not develop a VLS, only moderate increases in C3a levels (up to 13 nmol/liter) were observed. The highest levels at the first day of the regimen (mean: 7 nmol/liter) occurred 8 h after the IL-2 infusion. Thus, administration of IL-2 induces a dose-dependent activation of the complement system in vivo, which appeared to be related to the development of side effects of this therapy, such as the VLS.
Requirement for B cells for activation of contrasuppressor T cells by type III pneumococcal polysaccharide.
Authors: Braley-Mullen H
Published: December 2022

Type III pneumococcal polysaccharide (S3) is unable to activate S3-specific contrasuppressor T cells (Tcs) in mice depleted of B cells by chronic anti-IgM treatment or in immune defective xid mice that lack the B cell subset required for anti-S3 antibody responses. The inability of S3 to activate Tcs in xid mice was shown to be due to a requirement of B cells for Tcs activation rather than to an absence of Tcs in xid mice. The B cells from normal mice that are required for Tcs activation apparently function to present the S3 Ag to Tcs. S3 physically coupled to spleen cells (S3-SC) prepared from normal BCF1 SC could activate Tcs in both xid and BCF1 mice whereas S3-SC prepared from xid SC or B cell-depleted BCF1 SC could not activate Tcs in either strain. B cell APC function was abrogated by 3000 R irradiation and by treatment of the B cells with either chloroquine or paraformaldehyde. Interestingly, B cells from mice previously immunized with S3 were unable to function in Tcs activation; preimmunization of B cell donors with an irrelevant Ag or with a T-dependent form of S3 had no effect on their ability to function as APC. These latter observations are discussed in terms of the in vivo persistence of polysaccharide Ag and their ability to induce B cell tolerance under the experimental conditions used for these experiments. The results of this study provide evidence that B cells play an important and apparently obligatory role in the activation of Tcs by S3; B cells apparently function to present Ag to Tcs, resulting in the activation of this regulatory T cell subset.
Stimulation of B10.BR T cells with superantigenic staphylococcal toxins.
Authors: Callahan J E , Herman A , Kappler J W , Marrack P
Published: December 2022

The Staphylococcus aureus enterotoxins are known to be potent T cell activators, stimulating cell proliferation and lymphokine production. Two additional S. aureus proteins, exfoliating toxin and toxic shock syndrome toxin, share these properties. Recently these molecules have been termed "super-antigens" because of their ability to bind to class II MHC molecules and thus form ligands that interact with TCR in an unconventional manner. In this paper we show that each toxin stimulates mouse T cells bearing receptors that include particular V beta regions, almost regardless of the other variable receptor components. In addition, different toxins have different specificities for V beta.
Genetic analysis of the presentation of minor lymphocyte-stimulating determinants. II. Differing non-MHC control of super-stimulatory and more poorly stimulatory Mls phenotypes.
Authors: Ryan J J , LeJeune H B , Mond J J , Finkelman F D
Published: December 2022

Analysis of the capacity of splenocytes from non-prototypic Mlsa or Mlsc mouse strains to stimulate allogeneic H-2k-compatible T cells in a primary Mls-defined MLR provided interesting examples of exceptions to the usually stated characterization of Mlsa and Mlsc determinants as highly stimulatory of weakly stimulatory, respectively. Across the Mlsa barrier, MA/My stimulator cells had a significantly reduced capacity to elicit responder proliferation in comparison with prototypic AKR/J or less well studied C58/J, CE/J, or RF/J splenocytes. Across the Mlsc barrier, a gradient of stimulatory ability was observed with RF/J splenocytes being virtually nonstimulatory, prototypic C3H/HeJ splenocytes having an intermediate capacity, and CE/J and C58/J being highly stimulatory presenters of this non-MHC specificity. The differing capacity of each of these H-2k stimulator cells to elicit unprimed responder cell proliferation across an Mlsa or Mlsc difference correlated with the T cell growth factor activity that was secreted into the MLR supernatants. The super stimulatory form of Mlsc was expressed in an autosomal dominant fashion by (Mlsc poorly stimulatory x Mlsc super-stimulatory)F1 animals, (BALB.K x C58/J)F1 or (RF/J x CE/J)F1. The segregation of Mlsc stimulatory ability among first backcross and F2 animals derived from the former F1 was compatible with a single non-MHC gene controlling the expression and presentation of the super-stimulatory form of Mlsc. The regulatory nature of this gene was indicated by the observation that F1 animals generated from the Mlsc nonprototypic and poorly stimulatory BALB/c parental strain were self-tolerant to the super-stimulatory form of Mlsc. The existence of an Mls specificity other than a and c was suggested by positive non-MHC MLR responses in certain responder/stimulator cell combinations of Mls prototypic and nonprototypic mouse strains.
Lymphocyte proliferative responses to soluble and liposome-conjugated envelope peptides of HIV-1.
Authors: Krowka J , Stites D , Debs R , Larsen C , Fedor J , Brunette E , Düzgünes N
Published: December 2022

The proliferation of lymphocytes from HIV-seronegative (HIV Ab-) and seropositive (HIV Ab+) individuals in response to two synthetic peptide epitopes of HIV envelope glycoproteins (ENVgp) was evaluated as an index of cell-mediated immunity in infected individuals. All HIV Ab- and most HIV Ab+ individuals' lymphocytes failed to proliferate in primary cultures in response to the two soluble HIV ENVgp peptides, ENVP346 and ENVP466 even in the presence of rIL-2. After stimulation with liposome-conjugates of ENVP346 or ENVP466 and soluble rIL-2, however, CD4 lymphocytes from some HIV Ab+ individuals were able to proliferate. Significantly higher frequencies of rIL-2-augmented proliferative responses to liposome-conjugated ENVP346 or ENVP466 were observed in HIV Ab+ asymptomatic individuals as compared to patients with AIDS-related conditions or AIDS. These studies indicate that the conjugation of HIV peptides or proteins to liposomes and stimulation with rIL-2 may enhance cell-mediated responses to these peptides.
Cytokine-induced gene expression of a neutrophil chemotactic factor/IL-8 in human hepatocytes.
Authors: Thornton A J , Strieter R M , Lindley I , Baggiolini M , Kunkel S L
Published: December 2022

The liver participates in inflammation via the elaboration of acute phase proteins from hepatocytes in response to IL-1, TNF-alpha, and IL-6/INF-beta 2/hepatocyte-stimulating factor. In addition, some inflammatory states of the liver are characterized by leukocyte infiltrates. Here we demonstrate that human hepatocyte lines are capable of expressing mRNA and biologic activity for a neutrophil chemotactic factor (NCF)/IL-8 in response to the inflammatory mediators IL-1 alpha, IL-1 beta, and TNF. Two human hepatoma cell lines (SK-Hep and Hep-G2) displayed a time- and dose-dependent increase in steady state levels of NCF/IL-8 mRNA and secretion of chemotactic activity in response to TNF and IL-1. Neutralizing antibody to NCF/IL-8 inhibited hepatocyte-derived chemotactic activity by 88%. In contrast to IL-1 and TNF, hepatocytes did not respond to LPS or IL-6 within the time and dose parameters used above. Although the expression of NCF/IL-8 mRNA (1.8 kb) was first detectable between 1 and 2 h poststimulation, significant chemotactic bioactivity was not observed until about 4 h. Heat-inactivated (100 degrees C, 30 min) cytokine failed to induced NCF/IL-8 mRNA synthesis, and cotreatment of cells with cytokine and cycloheximide super-induced NCF/IL-8 mRNA while inhibiting production of bioactivity. Thus, NCF/IL-8 expression is a primary induction phenomenon. Our data demonstrate the stimulus specific induction of NCF/IL-8 in hepatocytes and suggest that cytokine cell-to-cell communication circuits may be important in neutrophil-mediated inflammatory processes in the liver.
Diverse VH and VL genes are used to produce antibodies against a defined protein epitope.
Authors: Andria M L , Levy S , Benjamini E
Published: December 2022

mAb secreting hybridomas were produced from mice hyperimmune to the model Ag tobacco mosaic virus protein. Six mAb were selected for their ability to bind synthetic peptides corresponding to amino acid residues 103-112 and 97-107 of tobacco mosaic virus protein. These mAb were analyzed for their fine specificity by measuring binding to synthetic analogs of the decapeptide, and cDNA sequences encoding the mAb V regions were determined. These analyses revealed that a wide range of different V regions are capable of binding with the same decapeptide epitope, and that these antibody sequence differences generally coincided with different binding fine specificities. This diverse antibody response with specificity for the same epitope demonstrates both the breadth of potential of the immune system and the lack of exclusivity in specific protein:protein interactions.
A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus.
Authors: Weisbart R H , Noritake D T , Wong A L , Chan G , Kacena A , Colburn K K
Published: December 2022

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.