bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement of thiol compounds as reducing agents for IL-2-mediated induction of LAK activity and proliferation of human NK cells.
Authors: Yamauchi A , Bloom E T
Published: December 2022

Thiol-related compounds, such as L-cystine, 2-ME or reduced glutathione (GSH), are important in many lymphoid cell activation pathways. We investigated their role in IL-2-generated lymphokine-activated killer (LAK) activity in human NK (CD16+ and/or CD56+) cells. Depletion of GSH and L-cystine, but not L-leucine or glycine, from medium used during culture of NK cells with IL-2 inhibited LAK and proliferative activities, whereas IL-2-independent lytic function of NK cells remained intact. Addition of L-cysteine, 2-ME or GSH, but not methylated analogs of these compounds (which cannot function as proton donors) to L-cystine/GSH-depleted medium restored proliferative response of NK cells and LAK generation. In the presence of L-buthionine-(S,R)-sulfoximine, an inhibitor of GSH synthesis, IL-2-induced LAK activity and proliferation of NK cells in medium without L-cystine and GSH, could be restored, at least in part, by addition of GSH, but not 2-ME or L-cystine. Furthermore, intracellular GSH levels were depressed in cells cultured in L-cystine/GSH-depleted medium but could be restored by the addition of 2-ME. The results suggest that (1) L-cystine or thiol containing compounds such as L-cysteine, 2-ME, or GSH are necessary for effective IL-2-activation of human NK cells, (2) these compounds must be functional proton-donors, i.e., reducing agents, implying regulation of the IL-2 activation pathway by oxidation-reduction, and (3) GSH synthesis is necessary for the activation. Experiments were performed to begin to dissect the redox-sensitive step(s) in LAK development. Depletion of reducing agents had no effect on internalization of rIL-2. In contrast, intracellular granzyme A activity was significantly depressed in NK cells cultured with rIL-2 in L-cystine/GSH-depleted medium compared with those cultured in medium in which L-cystine levels had been replenished. The findings suggest that step(s) in the transduction of the IL-2 signal in NK cells, between the internalization of IL-2 and the maturation of the lytic mechanism, are subject to regulation by oxidation-reduction.
Dissociation of IL-1 beta synthesis and secretion in human blood monocytes stimulated with bacterial cell wall products.
Authors: Chin J , Kostura M J
Published: December 2022

Human blood monocytes synthesize but do not secrete IL-1 beta in response to low doses of bacterial cell-wall products. With this observation, we have developed a two-step staged release assay that separates IL-1 beta synthesis from secretion. This assay can be used to identify secretagogues or inhibitors of IL-1 beta secretion as well as the biochemical events leading to IL-1 beta release. Human blood monocytes are first treated with low (< 50 pg/ml) doses of LPS, which causes the synthesis of intracellular proIL-1 beta. Release of intracellular IL-1 beta can be induced by further treatment with 100 ng/ml of LPS or 1 x 10(6) CFU/ml of heat-killed Staphylococcus aureus. The amount and the efficiency of IL-1 beta secretion in the staged release assay was comparable with that of a standard method of treating blood monocytes with a single dose of 100 ng/ml LPS. Ongoing protein synthesis was not required for IL-1 beta secretion because mature IL-1 beta release occurred in the presence of the protein synthesis inhibitor cycloheximide. We have compared the effects of four different inhibitors of cytokine synthesis on IL-1 beta production in the standard and staged release assays. We find that dexamethasone or IL-10, when added together with 100 ng/ml LPS, inhibits IL-1 beta production with IC50 levels of 0.2 microM and 2.0 ng/ml, respectively. The IC50 levels increase greater than 50-fold when tested against monocytes pretreated with 50 pg/ml of LPS. These data suggest that dexamethasone and IL-10 have little effect on IL-1 beta secretion. Conversely, the IL-1 beta converting enzyme inhibitor, Ac-Tyr-Val-Ala-Asp-CHO (L-709,049) and the anti-inflammatory agent [5-(4-pyridyl)6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b)thiazole] (SK&F 86002) inhibited IL-1 beta release in both the standard and staged release assays with IC50 of 1 microM. The data suggest that L-709,049 and SK&F 86002 interfere with steps involved in IL-1 beta secretion, as opposed to synthesis. The results also document a novel method for delineating separate events in the pathway required for IL-1 beta biosynthesis and further distinguish two classes of compounds capable of modulating IL-1 beta secretion.
Antigen and carbachol mobilize calcium by similar mechanisms in a transfected mast cell line (RBL-2H3 cells) that expresses ml muscarinic receptors.
Authors: Choi O H , Lee J H , Kassessinoff T , Cunha-Melo J R , Jones S V , Beaven M A
Published: December 2022

Because of unresolved questions about the mechanism of Ag-stimulated Ca2+ influx, Ca2+ mobilization in response to carbachol and Ag was compared in transfected rat basophilic RBL-2H3(ml) cells that expressed both Fc epsilon and ml muscarinic receptors. Although the stimulants activated phospholipase C via different coupling mechanisms, a G protein for carbachol or a tyrosine kinase for Ag, they released Ca2+ from the same intracellular pool and used the same or very similar mechanisms for influx of Ca2+ as indicated by the similar patterns of inhibition of uptake of 45Ca2+ by various cations. With both stimulants, influx and sustained increases in free cytosolic Ca2+ ([Ca2+]i) were associated with relatively small increases in inositol 1,4,5-trisphosphate (IP3). Blockade of Ca2+ influx resulted in rapid decline in [Ca2+]i to basal levels; resumption of influx caused a substantial "spike" in [Ca2+]i before [Ca2+]i reequilibrated at the same former steady-state levels but without perturbing levels of IP3. Thus, the refilling and discharge of Ca2+ from IP3-sensitive stores might occur synchronously on resumption of influx, or asynchronously during sustained influx and elevation of [Ca2+]i. Together, the results suggested that influx of Ca2+ in response to stimulation via Fc epsilon receptors occurred through a pathway, analogous to that observed with other types of stimulants, in which Ca2+ influx follows emptying of intracellular Ca2+ stores by IP3. Also, secretion was highly dependent on this IP3-dependent pathway.
Increased synthesis and secretion of a 14-kDa phospholipase A2 by guinea pig alveolar macrophages. Dissociation from arachidonic acid liberation and modulation by dexamethasone.
Authors: Hidi R , Vargaftig B B , Touqui L
Published: December 2022

The occurrence of a 14-kDa secretory phospholipase A2 (PLA2) in guinea pig alveolar macrophages (AM) and its relationship with the release of arachidonic acid (AA) were investigated. Freshly collected AM showed no detectable PLA2 activity as measured by the in vitro hydrolysis of phosphatidic acid. However, the PLA2 activity increased progressively when AM were maintained in culture to reach a level 60- to 100-fold greater than basal values within 20 h, with a parallel secretion into the incubation medium. By contrast, the activities of other phospholipid-hydrolyzing enzymes (platelet-activating factor acetylhydrolase and lysophospholipase) were modified only marginally. Both intra- and extracellular increases of PLA2 activity were abrogated with actinomycin D or cycloheximide. The enhanced PLA2 activity preferentially hydrolyzed negatively charged phospholipids in the order phosphatidic acid > phosphatidylglycerol > phosphatidylethanolamine > phosphatidylcholine, had an optimum pH of 7.5, and required a millimolar Ca2+ concentration for optimal activity and an apparent molecular mass of 14 kDa. Taken together, these results suggest that cultured AM elaborate an enzyme similar to the group II PLA2. On the other hand, our results show that AM hydrolyzed exogenous 2-arachidonoyl phosphatidylcholine and released AA and metabolites on FMLP stimulation. However, in contrast to the increase observed in the activity of the 14-kDa PLA2, the enzymatic activity involved in the hydrolysis of 2-arachidonoyl phosphatidylcholine and AA release remained constant with the culture duration of AM. Finally, dexamethasone markedly inhibited the increase of PLA2 activity, but only marginally inhibited the release of AA and metabolites from FMLP-stimulated AM. We conclude that guinea pig AM elaborate a 14-kDa PLA2 similar to the group II PLA2 through RNA- and protein synthesis-dependent processes. This elaboration appears to be induced by the adhesion of AM and is clearly dissociated from the liberation of AA.
Protective effects of IL-4 and IL-10 against immune complex-induced lung injury.
Authors: Mulligan M S , Jones M L , Vaporciyan A A , Howard M C , Ward P A
Published: December 2022

Recombinant murine IL-4 and IL-10 have been used in two models of inflammatory lung injury in rats after intrapulmonary deposition of IgG or IgA immune complexes. These models have contrasting requirements for cytokines, phagocytic cells, and adhesion molecules. In these two models of lung injury, IL-4 and IL-10 were individually coinstilled into the airways with the IgG or IgA antibodies, whereas the Ag were injected intravenously. Injury was quantitated by increases in permeability (leakage of 125I-BSA) and by hemorrhage (extravasation of 51Cr-RBC). In the model of IgG immune complex-induced lung injury, IL-4 and IL-10 were each highly protective when given in nanogram amounts. These protective effects were dose dependent. IL-4 and IL-10 caused substantial reductions in lung content of myeloperoxidase and parallel reductions in neutrophil content in bronchoalveolar lavage (BAL) fluids. The protective effects of IL-4 and IL-10 were associated with profound reductions of TNF-alpha in the BAL fluids and complete inhibition in the up-regulation of pulmonary vascular ICAM-1. In the IgA immune complex model of lung injury IL-4 had no protective effects, whereas IL-10 was highly protective. These protective effects correlated with diminished retrieval of alveolar macrophages in BAL fluids. These data suggest that IL-4 and IL-10 have significant protective effects in lung inflammatory injury, presumably achieving these effects by various mechanisms.
Autoimmunity and filariasis. Autoantibodies against cytoplasmic cellular proteins in sera of patients with onchocerciasis.
Authors: Meilof J F , Van der Lelij A , Rokeach L A , Hoch S O , Smeenk R J
Published: December 2022

Onchocerciasis or "river blindness" is a vector-borne tropical disease resulting from infection with the filarial nematode Onchocerca volvulus. Disease manifestations include dermatitis, rheumatic complaints, and blindness. Recent findings have suggested an autoimmune etiology for the occurrence of chorioretinopathy, a disease of the eye which together with sclerosing keratoconjunctivitis is responsible for approximately 400,000 onchocerciasis-related cases of blindness. The identification of onchocerciasis as an important cause of tropical rheumatism prompted us to evaluate serologically the presence of systemic autoimmune disease in onchocerciasis patients and local controls from a hyperendemic area in Sierra Leone. In both groups there was a marked autoimmune response against cytoplasmic non-RNA-associated proteins consisting of autoantibodies against five major Ag with respective m.w. of 35, 51, 64, 83, and 110 kDa. These five proteins are novel autoantigens that could be distinguished from calreticulin, the human homologue of the onchocercal Ag RAL-1, and known autoantigens such as the 50-kDa La/SS-B or 52- and 60-kDa Ro/SS-A proteins by immunoblotting and ELISA assays. Furthermore, autoantibody reactivity against calreticulin was significantly higher in O. volvulus-infected individuals than in endemic controls. Autoantibody reactivity against the five major autoantigens, anti-calreticulin reactivity, and antibody reactivity against the 65-kDa arthritis-associated mycobacterial heat shock protein were intercorrelated as parts of an onchocerciasis-associated autoimmune response. The implication of autoimmunity in the disease pathogenesis of onchocerciasis could have important consequences for future research on therapeutical regimens, pathogenetic mechanisms, and serological diagnosis of onchocerciasis.
Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes.
Authors: Geng Y , Zhang B , Lotz M
Published: December 2022

Bacterial LPS induce production of cytokines such as IL-1, IL-6, and TNF in mononuclear phagocytes, and this represents a central component in the pathogenesis of septic shock syndrome. However, the mechanisms by which LPS activates these cells to express cytokines are not completely characterized. The present study addressed the role of different protein kinases in the LPS induction of cytokines. It is shown that LPS induced a 12- to 16-fold increase in IL-1 beta, IL-6, and TNF-alpha mRNA levels, and this was completely or more than 80% blocked by the protein tyrosine kinase specific inhibitors herbimycin A and genistein at the concentrations of 1.7 and 37 microM, respectively. Protein kinase C inhibition by staurosporine reduced LPS induction of TNF-alpha, whereas it had no effects on IL-6 and IL-1 beta. Inhibition of protein kinase A by H89 reduced IL-6 mRNA levels but did not detectably change IL-1 beta or TNF-alpha mRNA levels. In contrast, LPS did not increase leukemia inhibitory factor mRNA, which was constitutively expressed and not significantly reduced by these inhibitors. In addition to cytokine mRNA levels, LPS-induced IL-6 protein synthesis and IL-6 bioactivity were also reduced to baseline levels by the PTK inhibitors herbimycin A and genistein. Both PTK inhibitors also reduced the LPS activation of nuclear factor-kappa B (NF-kappa B), which is a transcription factor involved in the expression of cytokine genes such as IL-6 and TNF-alpha. The activation of NF-kappa B was also reduced by H89, whereas staurosporine had no effect on this response. In summary, these findings suggest that protein kinase C and protein kinase A appear to have selective effects in the LPS induction of cytokines, whereas PTK is required for LPS induction of a broad spectrum of cytokines and NF-kappa B activation in monocytes.
Anti-CD11b antibody prevents immunopathologic changes in viable moth-eaten bone marrow chimeric mice.
Authors: Koo G C , Rosen H , Sirotina A , Ma X D , Shultz L D
Published: December 2022

The effect of in vivo treatment with anti-CD11b (MAC-1) antibody (Ab) was examined in an inflammatory disease model, the viable moth-eaten (mev) mutant mouse. The autosomal recessive mev gene occurred spontaneously as a point mutation of the hematopoietic cell protein tyrosine phosphatase in C57BL/6 mice. Homozygotes (mev/mev) develop a chronic myelomonocytic inflammation, involving accumulation of myelomonocytic cells in lungs and skin, resulting in interstitial pneumonitis and severe edema in the paws. These mice also exhibit abnormalities in lymphoid development, thymic atrophy, with T cell and NK cell dysfunction. These inflammatory changes are transferrable by bone marrow cells of mev/mev mice, indicating that mev mutation is due to a stem cell defect in the myelomonocytic pathway. An anti-CD-11b (5C6) Ab inhibited the immunopathologic changes in the bone marrow chimeras, when the Ab treatment was initiated on day -1 or day 0 of the bone marrow transplant. The lungs, paws, and thymus all remained normal after treatment. Furthermore, the Ab also delayed the onset of the mev syndromes when the Ab was given 10 days after the bone marrow transfer. Therefore anti-CD11b Ab inhibited inflammation both prophylactically and therapeutically, and restored normal function of T and NK cells in this disease model. These results support the contention that CD11b molecules expressed in the myelomonocytic cells play a critical role in this naturally occurring inflammatory disease.
Expression of GlyCAM-1, an endothelial ligand for L-selectin, is affected by afferent lymphatic flow.
Authors: Mebius R E , Dowbenko D , Williams A , Fennie C , Lasky L A , Watson S R
Published: December 2022

The interaction of naive, L-selectin-bearing lymphocytes with counterreceptors on the surface of high endothelial venules (HEV) is the initial step in the extravasation of these cells from the bloodstream into the peripheral lymph node. Recently, two sulfated glycoprotein ligands, 50 and 90 kDa, respectively, have been identified as ligands for L-selectin using an L-selectin-IgG chimera. cDNA cloning of one of these molecules, the 50-kDa sulfated glycoprotein (glycosylation-dependent cell adhesion molecule 1 [GlyCAM-1]), has shown it to be a mucinlike scaffold that presents a carbohydrate ligand(s) to the lectin domain of L-selectin. Herein, we analyze the factors that might regulate the expression of these ligands. Ligation of afferent lymphatics results in a complete loss of the mRNA for GlyCAM-1. In addition, L-selectin-mediated adhesion, as inferred by binding of an L-selectin-IgG chimera, is also lost on interruption of afferent flow. It thus appears that a soluble and/or cellular component(s) of afferent lymph regulates the expression of GlyCAM-1 mRNA and the resultant HEV adhesiveness for lymphocytes.
Isolation of a cDNA encoding thymic shared antigen-1. A new member of the Ly6 family with a possible role in T cell development.
Authors: MacNeil I , Kennedy J , Godfrey D I , Jenkins N A , Masciantonio M , Mineo C , Gilbert D J , Copeland N G , Boyd R L , Zlotnik A
Published: December 2022

We have previously characterized a novel mouse thymocyte marker, defined as thymic shared Ag-1 (TSA-1), present on both immature thymocytes and a subset of thymic medullary epithelial cells. MTS 35, a mAb specific for TSA-1, alters T cell differentiation when added to fetal thymic organ cultures, suggesting TSA-1 may be important for T cell development in the thymus. In this study, we describe the isolation of a cDNA encoding TSA-1 using transient expression of COS-7 cells and selection with MTS 35. The predicted amino acid sequence of this cDNA encodes a 15 to 17-kDa protein and the expressed protein is linked to the membrane via a phosphatidylinositol moiety. TSA-1 is transcriptionally active at various levels in all organs examined, suggesting that its role is not solely intrathymic. TSA-1 shares amino acid sequence homology to the mouse Ly6 multigene family, epidermal growth factor-like receptors, and to cobra venom neurotoxin. The Tsa-1 locus is located on chromosome 15 linked to Ly6 on the mouse genome. We also examined the effects of MTS 35 in fetal thymic organ cultures repopulated with two subsets of thymocytes representing defined stages of T cell development. Our results suggest that TSA-1 may play a role during positive selection and the transition from CD4+CD8+ thymocytes to the mature CD4+CD8- and CD4-CD8+ subsets.
Comparative analysis of the kinetics of binding and internalization of IL-5 in murine IL-5 receptors of high and low affinity.
Authors: Mita S , Takaki S , Tominaga A , Takatsu K
Published: December 2022

The high affinity IL-5R consists of at least two chains, alpha and beta. IL-5R alpha binds IL-5 with low affinity. IL-5R beta is required to construct the high affinity IL-5R, although IL-5R beta does not bind IL-5 by itself. To characterize the roles of IL-5R alpha and IL-5R beta on the association, dissociation, and internalization of IL-5, we compared the binding kinetics and the internalization of 35S-labeled IL-5 by high affinity IL-5R (dissociation constant approximately 150 pM) bearing T88-M with those by low affinity IL-5R (dissociation constant approximately 30 nM) bearing MOPC104E. We found that association kinetics of IL-5 to either natural receptor were similar. The maximal binding of IL-5 to both high and low affinity IL-5R was rapid (within 10 min). The dissociation of IL-5 from low affinity IL-5R was rapid (t1/2 < 30 min), but that from the high affinity IL-5R was remarkably slower (t1/2 > 120 min). The internalization of IL-5 was observed only in T88-M, but not in MOPC104E, suggesting that IL-5 internalization is mediated via high affinity IL-5R. Association and dissociation kinetics observed in natural cell line were mostly reproduced by the kinetic analysis of reconstituted IL-5R on transfectants with either the IL-5R alpha or the IL-5R alpha and IL-5R beta genes. However, transfectants that expressed IL-5R alpha significantly internalized IL-5, although the level was much lower than observed with IL-5R alpha beta transfectants. These results suggest that IL-5R alpha may be involved in the internalization of IL-5, whereas IL-5R beta is responsible for slowing the dissociation and the efficient internalization of IL-5 by stabilizing the ligand-receptor complex.
Characterization of an IgM Fc-binding receptor on human T cells.
Authors: Nakamura T , Kubagawa H , Ohno T , Cooper M D
Published: December 2022

The existence of an IgM receptor on human T cells has been suggested by T cell rosetting with IgM-coated erythrocytes. In this study we used immunofluorocytometry to demonstrate that 1) after short-term culture, a majority of the T cells can bind IgM at easily detectable levels, 2) human and mouse IgM preparations bind to human T cells but other Ig isotypes do not, 3) the IgM binding is saturable and inhibitable only by Ig of IgM isotype and 4) the Fc portion of IgM is involved in the binding to a protease-sensitive cell surface protein. Biochemical analysis of the T cell receptor for IgM reveals a cell surface protein of 60 kDa in comparison with the 58 kDa Fc mu receptors (Fc mu R) on B-lineage cells. Although Fc mu R expression is up-regulated after B cell activation, the reverse is true after T cell activation. In addition, the T cell Fc mu R is relatively resistant to phospholipase C treatment. These results indicate that T- and B-lineage cells express Fc mu R with different biologic characteristics.
Inhibitors of poly(ADP-ribose) polymerase increase antibody class switching.
Authors: Shockett P , Stavnezer J
Published: December 2022

Previous studies suggest that heavy chain isotype switch (S) recombination is directed by cytokine-induced transcription of the unrearranged CH gene before recombination. In studies aimed at identifying other signaling pathways that promote switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase LPS-induced switching to IgA in the B cell lymphoma 1.29 mu and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In 1.29 mu cells, PARP inhibitors increase IgA switching by day 2 and cause a fivefold increase in switching on day 3 as assayed by immunofluorescence microscopy. In spleen B cells, the PARP inhibitor nicotinamide increases IgG1 switching by about twofold. Nicotinamide also causes a reduced intensity of hybridization of C mu- and C alpha-specific probes to genomic DNA fragments containing the expressed VDJ-C mu and the unrearranged S alpha-C alpha segments, respectively, in 1.29 mu cells, indicating that PARP inhibition increases rearrangement of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogues or reduced by inhibitors of protein kinase A. Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged C alpha gene.
Substance P and somatostatin can modulate the amount of IgG2a secreted in response to schistosome egg antigens in murine schistosomiasis mansoni.
Authors: Blum A M , Metwali A , Mathew R C , Elliott D , Weinstock J V
Published: December 2022

Substance P (SP) and somatostatin 1-14 (SOM) have immunoregulatory properties. Cells within the granulomas of murine schistosomiasis mansoni make both. SP enhances, whereas SOM inhibits soluble egg Ag (SEA)-induced, IFN-gamma production. IFN-gamma is important during IgG2a isotype switching. Thus, we investigated whether SP or SOM could affect IgG2a production in murine schistosomiasis. Our results show that SEA and rIFN-gamma stimulate splenic IgG2a secretion in murine schistosomiasis. Moreover, SP at > or = to 10(-10) M substantially increased both polyclonal as well as SEA-specific, IgG2a secretion from spleen cells challenged with SEA. However, cells exposed to SOM at > or = 10(-10)M showed strong inhibition. Also, both SP and SOM modulated the frequency of IgG2a-producing cells. Splenic IgG2a production in response to SEA, SP, and SOM required the presence of Thy 1.2+ cells, whereas, rIFN-gamma- induced IgG2a synthesis did not. Also, experiments using irradiation lymphocytes showed that SP, SOM, or rIFN-gamma modulation of IgG2a release was not dependent on cell proliferation. The highly specific SP receptor antagonist, CP-96,345, completely inhibited the effect of SP but not SOM on IgG2a release. This suggests that SP acted through an authentic NK-1 receptor and that SOM required a different receptor interaction. Granuloma cells secreted IgG2a constitutively. Yet, neither SEA, SP, SOM, rIFN-gamma, nor blocking anti-IFN-gamma mAb could modulate this constitutive IgG2a release during short term culture conditions. Moreover, the IgG2a secretion also continued in the absence of Thy 1.2+ lymphocytes. However, mice treated with CP-96,345 or octreotide (SOM agonist) in vivo produced granulomas that made little or no IgG2a. Spleen cell experiments showed that SEA, SP, SOM, and rIFN-gamma could only affect SEA-induced, IgG2a production during early stages of Ag stimulation. Thus, unlike the spleen, it is probable that the granulomas contain mostly activated B cells that have completed switch recombination.
Inhibition of C3 and IgG proteolysis enhances phagocytosis of Porphyromonas gingivalis.
Authors: Cutler C W , Arnold R R , Schenkein H A
Published: December 2022

In the face of an apparently competent immune response to Porphyromonas gingivalis, it is unclear how P. gingivalis evades the immune response and persists in human periodontitis. Particularly germane may be its ability to resist phagocytosis by degrading and not binding serum opsonins. In our study, the resistance by invasive (W83 and A7436) and noninvasive (ATCC 33277) P. gingivalis strains to phagocytosis by human neutrophils was compared with their C3- and IgG-proteolytic activity. The ability of opsonic human serum antibody to inhibit C3 proteolysis was also evaluated. Our results indicate that the more phagocytosis-resistant invasive strains accumulate less 125I-C3 than the noninvasive strain; moreover, invasive strains degrade complement C3 in a dose-dependent manner, inhibitable by rabbit antiserum or adult periodontitis serum. Opsonization and C3 accumulation on strain A7436 were both facilitated by pretreatment with rabbit antiserum, certain adult periodontitis sera, protease inhibitors (p-chloromercuriphenylsulfonic acid, N alpha-p-tosyl-L-lysine chloromethyl ketone, diisopropylfluorophosphate), heat (60 degrees C, 15 min), and were Mg2+ dependent. The sera from 13 human subjects with or without periodontitis were assayed for antibody titers to P. gingivalis (ELISA units), opsonic activity (% of PMN engaged in phagocytosis) and enhancement of C3 accumulation. Statistically significant associations were observed between % of PMN engaged in phagocytosis and % C3 accumulation, between % of PMN engaged in phagocytosis and ELISA units and between % C3 accumulation and ELISA units. Degradation of purified rabbit IgG, but not specific antibody-containing rabbit IgG by P. gingivalis A7436 was observed, and was inhibited by diisopropyl fluorophosphate (DFP) or cold (2 degrees C). Our data suggest that C3 and IgG cleavage by P. gingivalis proteases are inhibitable by antibody and are contributory factors in, but are not the sole determinants of, phagocytosis resistance.
T cells bearing V beta 6 T cell receptors in the cell-mediated immune response to Mycobacterium leprae.
Authors: Wang X , Golkar L , Uyemura K , Ohmen J D , Villahermosa L G , Fajardo T T , Cellona R V , Walsh G P , Modlin R L
Published: December 2022

The skin lesions of leprosy provide a window into the immunoregulatory events involved in the human immune response to infection. T cells are thought to play a vital role in the pathogenesis of different forms of the disease. To identify predominant specific T cell subpopulations in leprosy lesions, the TCR-beta chain repertoire was simultaneously studied in skin biopsy specimens and PBMC from both immunologically resistant tuberculoid leprosy and susceptible lepromatous leprosy patients. This was accomplished by obtaining RNA from lesions and PBMC, synthesizing cDNA, and performing the polymerase chain reaction. We found that TCR gene subfamilies V beta 6.1 through V beta 6.4 (V beta 6.1-4) were strikingly overrepresented in lesions vs PBMC of seven of nine tuberculoid patients but only one of nine lepromatous patients. Similarly, V beta 6.5/6.8/6.9 subfamilies were predominant in four of nine tuberculoid patients, but none of the nine lepromatous patients. To explore the influence of the complementarity-determining region 3 (CDR3) in selection of T cells expressing V beta 6 TCR, we sequenced the V beta 6.1-4-C beta polymerase chain reaction products derived from the lesions and PBMC of two tuberculoid patients. From the analysis of deduced amino acid sequences, we found conserved amino acid residues and amino acid motifs in the CDR3 region of the lesion-derived sequences from each patient. Our data suggest that the nominal Ag select T cells bearing V beta 6 TCR in the cell-mediated immune response to Mycobacterium leprae.
Induction of porcine granulocyte-mediated tumor cytotoxicity by two distinct monoclonal antibodies against lytic trigger molecules (PNK-E/G7).
Authors: Wierda W G , Johnson B D , Dato M E , Kim Y B
Published: December 2022

PNK-E and G7 mAb bind to distinct porcine granulocyte function-associated molecules and induce granulocyte-mediated cytotoxicity against tumor targets. PNK-E mAb binds to a 205-kDa molecule that reduces to a dispersed band of 50 kDa on SDS-PAGE analysis. Previous work demonstrates that G7 mAb immunoprecipitates a molecule that appears as a heterodispersed 40-kDa band under both reducing and nonreducing conditions on SDS-PAGE analysis. Whole but not F(ab')2 PNK-E and G7 mAb induce dose-dependent porcine granulocyte-mediated lysis of FcR+ but not FcR- targets, suggesting a redirected cytotoxicity mechanism of granulocyte-mediated killing. Fresh porcine granulocytes also mediate significant levels of antibody-dependent cellular cytotoxicity (ADCC) against nucleated (SB) target cells. Neither whole nor F(ab')2 PNK-E mAb affects granulocyte-mediated ADCC against SB target cells. However, both whole and F(ab')2 G7 mAb inhibit granulocyte-mediated ADCC against SB targets by approximately 50%. Bound F(ab')2 G7 mAb inhibits PNK-E mAb-induced granulocyte-mediated cytotoxicity against K562 targets, but bound F(ab')2 PNK-E mAb does not inhibit G7 mAb-induced granulocyte-mediated cytotoxicity, suggesting a physical association between the PNK-E and G7 molecules on the surface of porcine granulocytes. PNK-E and G7 hybridoma cells are readily lysed by granulocyte effectors, further supporting that the PNK-E and G7 molecules are cytolytic trigger molecules on granulocytes. These data demonstrate that PNK-E and G7 mAb bind to distinct granulocyte lytic trigger molecules and induce potent granulocyte-mediated cytotoxicity against nucleated tumor targets through a mechanism of redirected cytotoxicity.
Combined anti-CD2 and anti-CD3 receptor monoclonal antibodies induce donor-specific tolerance in a cardiac transplant model.
Authors: Chavin K D , Qin L , Lin J , Yagita H , Bromberg J S
Published: December 2022

Administration of mAb against either the CD2 or CD3 receptor prolongs graft survival in CBA recipients in a heterotopic, nonvascularized cardiac transplant model, whereas the combination of mAb produces indefinite survival. Combined alpha-CD2 plus alpha-CD3 mAb synergistically prolonged allograft survival indefinitely for C57BL/6 donor hearts (> 150 vs 13.4 +/- 0.5 days for controls, p < 0.001, Wilcoxon's sign rank). All second donor-specific C57BL/6 allografts survived > 100 days (p < 0.001) without any additional immunosuppression. Third-party BALB/c allografts were rejected in a first set fashion (14.2 +/- 0.5 days). Anti-CD2 mAb of other epitopic specificities and isotypes demonstrate equivalent immunosuppressive capacity. The combination of mAb resulted in indefinite graft survival in other strain combinations. Therefore, these results are not restricted to a particular alpha-CD2 mAb or MHC combination. Combinations of alpha-CD2 plus mAb with specificities other than to CD3 did not result in tolerance, showing that the CD2-CD3 interaction was critical for tolerance induction. CTL and MLR responses from tolerant animals were normal both to H-2b and H-2d stimulators, indicating that clonal deletion of effector T cells did not occur. Adoptive transfer of naive recipient type cells broke tolerance, showing that graft adaptation was not the major determinant of tolerance maintenance. Flow cytometric analysis demonstrated that tolerance was not associated with deletion of T cells. The results imply that the mechanism of tolerance induction is related to suppression and/or anergy of helper and effector cells at the time of allografting, whereas maintenance of tolerance is associated with anergy in the Th cell compartment.
Immortalization of human T cell clones by Herpesvirus saimiri. Signal transduction analysis reveals functional CD3, CD4, and IL-2 receptors.
Authors: Bröker B M , Tsygankov A Y , Müller-Fleckenstein I , Guse A H , Chitaev N A , Biesinger B , Fleckenstein B , Emmrich F
Published: December 2022

Investigation of human activated T cells has been complicated by the need for periodic restimulation with Ag/mitogen and accessory cells and by the limited life span of most human T cell clones. To overcome these problems, we have transformed established human T cell clones to permanent growth with Herpesvirus saimiri, a lymphoma-inducing virus of nonhuman primates. Three human CD4+ T cell clones were investigated in detail. They have been growing in the presence of exogenous IL-2 but without restimulation with mitogen or feeder cells for more than 11 mo with doubling times between 2 and 4 days. In contrast, their nontransformed parent clones needed to be restimulated with PHA and feeder cells every 14 to 21 days. To compare responses of H. saimiri-transformed clones with those of their parent clones, we stimulated the cells with IL-2 or with anti-CD3 and/or anti-CD4 mAb with and without cross-linking on the cell surface. Transformed and nontransformed T cell clones were strikingly similar in parameters of early signal transduction, namely, tyrosine phosphorylation and mobilization of calcium. Ligation of their TcR/CD3 complexes by mAb or by Ag in the presence of autologous accessory cells increased the proliferation and the secretion of IFN-gamma. Taken together, we have shown that human T cell clones immortalized with H. saimiri express functional CD3, CD4, and IL-2R. They constitute a simple, stable, reproducible and accessory cell-free model system for the investigation of signal transduction events in activated human T cells.
A conserved binding site on the receptor for polymeric Ig is homologous to CDR1 of Ig V kappa domains.
Authors: Bakos M A , Kurosky A , Czerwinski E W , Goldblum R M
Published: December 2022

We have previously shown that the binding of human secretory component (SC) to polymeric IgA (PIgA) is inhibited by a synthetic peptide fragment of SC (SC[15-37]), and also by a mAb (mAb 6G11) which specifically recognizes the unbound form of SC and SC[15-37]. In this report we show that the binding of bovine, rabbit, and rat SC to human PIgA can also be inhibited by SC[15-37], and that the relative affinity of these three species of SC for human PIgA correlated with their relative affinity for mAb 6G11. The binding of mAb 6G11 to SC isolated from various species was especially affected by amino acid substitutions at position 26 of SC. Structural modeling revealed that the side chain of residue 26 is located on the external face of a loop in SC domain I which is homologous in size and position to the first complementarity determining region (CDR1) of Ig V kappa domains. However, the surface topography of the CDR1-like loop of SC is distinct from other known V kappa CDR1 loops due to the unique distribution of charged and bulky polar side chains which locate to the exterior face of the loop. Together, these findings indicated that the binding site on the receptor for PIg is highly conserved among species, and is composed, in part, of residues of domain I of SC which may form a distinct CDR1-like loop on the surface of the molecule.
Analysis of cytokine mRNA expression during primary granuloma formation induced by eggs of Schistosoma mansoni.
Authors: Wynn T A , Eltoum I , Cheever A W , Lewis F A , Gause W C , Sher A
Published: December 2022

Granulomas induced by parasite eggs are the primary lesions in mice infected with Schistosoma mansoni. Temporal analysis of cytokine mRNA expression in lung tissue containing synchronized granulomas demonstrated a Th0-like pattern of lymphokine expression. IFN-gamma, IL-1 beta, and IL-6 were the primary cytokines induced by day 1 in developing lung granulomas initiated by i.v. egg injection. These changes were followed by increases in expression of IL-2, IL-4, and IL-10 mRNA on day 3 and TNF-alpha and IL-5 mRNA on day 6. Nearly all cytokine mRNA reached maximal levels by day 6, which preceded the peak in granuloma size seen on day 14. In vivo treatment of egg-injected mice with either anti-IL-2 or anti-IL-4 antibodies significantly diminished the size of circumoval granulomas in the lungs. Both groups of antibody-treated animals displayed a marked reduction in IL-4 as well as IL-5 mRNA expression, although IFN-gamma and IL-2 mRNA levels were unchanged or slightly increased. These findings confirm previous observations suggesting a role for IL-2 in egg-induced pathology via the generation of Th2-associated responses, and also indicate a primary function for IL-4 in granuloma formation. Analysis of responses after injection of eggs into nude mice demonstrated that only the Th2 cytokines IL-4 and IL-5 are exclusively dependent on T cells for their induction. Taken together, these data suggest that Th2 cells producing IL-4 play a major role in egg granuloma formation, and that the induction and ultimate down-modulation of Th2-like responses may be influenced by non-T-cell-derived cytokines.
Orthovanadate induces translocation of phospholipase C-gamma 1 and -gamma 2 in permeabilized mast cells.
Authors: Atkinson T P , Lee C W , Rhee S G , Hohman R J
Published: December 2022

Rapid activation of phospholipase C (PLC) with a resultant increase in phosphatidylinositol hydrolysis occurs after aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells. We previously described an increase in PLC activity associated with the particulate fraction of rat basophilic leukemia (RBL) cells after Fc epsilon RI aggregation, and this redistribution of enzyme activity correlated with an increase in immunoreactivity of the gamma 1 isozyme of PLC in the particulate fraction by Western blot analysis (J. Immunol. 148:2194-2200, 1992). We now report that the tyrosine phosphatase inhibitor orthovanadate mimics Fc epsilon RI-mediated activation of PLC-gamma 1 in RBL cells after permeabilization with Staphylococcus aureus alpha-toxin. Orthovanadate treatment of permeabilized cells induced: 1) a large increase in phosphoinositide hydrolysis in endogenously labeled cells; 2) an increase in PLC activity associated with the particulate fraction; and 3) an increase in immunoreactivity of PLC-gamma 1 in Western blots of the particulate fraction. In addition, incubation of RBL cells with either oligomeric IgE or orthovanadate results in the translocation of PLC-gamma 2 from the cytosol to the particulate fraction. All of the above effects were qualitatively similar to those seen after Fc epsilon RI aggregation. These data suggest that translocation and activation of PLC in mast cells are controlled by tyrosine phosphorylation of either the enzyme itself or some regulatory component. The equilibrium can be shifted to the phosphorylated state during either receptor-mediated activation of a tyrosine kinase or by blockade of dephosphorylation.
Phagocytosis of Staphylococcus aureus and Haemophilus influenzae type B opsonized with polyclonal human IgG1 and IgG2 antibodies. Functional hFc gamma RIIa polymorphism to IgG2.
Authors: Bredius R G , de Vries C E , Troelstra A , van Alphen L , Weening R S , van de Winkel J G , Out T A
Published: December 2022

To assess the function of IgG subclass antibodies we studied the opsonization of Staphylococcus aureus (STAW) and Haemophilus influenzae type b (Hib) by natural IgG1 and IgG2 antibodies from five healthy individuals. Phagocytosis by polymorphonuclear leukocytes (PMN) was analyzed using FITC-labeled bacteria and flow cytometry. All PMN donors were typed for the High- and Low-Responder phenotype of the human Fc gamma receptor (hFc gamma) type IIa (CD32) and the NA1/NA2 allotype of the hFc gamma RIIIb (CD16). When PMN were used that were heterozygous for hFc gamma RIIa and hFc gamma RIIIb, phagocytosis of STAW opsonized with IgG1 antibodies was similar to that with IgG2, both in the presence and absence of a source of complement (agammaglobulinemic serum). Phagocytosis of Hib opsonized with IgG2 anti-Hib proved significantly lower (p < 0.05) than with IgG1 anti-Hib using preparations from 4 of the 5 individuals tested. IgG2 anti-Hib from one donor, however, proved more effective than IgG1 anti-Hib. The properties of PMN with hFc gamma RIIaLR,LR and hFc gamma RIIaHR,HR were compared, using hFc gamma RIIIb NA1/NA2-matched PMN. hFc gamma RIIaHR,HR PMN were virtually incapable of phagocytosing STAW and Hib opsonized with IgG2 anti-bodies without complement, in contrast to hFc gamma RIIaLR,LR PMN. Phagocytosis of IgG2-oposonized bacteria by hFc gamma RIIaLR,LR PMN was effectively inhibited by mAb against hFc gamma RIIa (IV.3). IgG1-mediated phagocytosis was blocked by mAb against hFc gamma RIIIb (CLB/FcRGran 1) to a greater extent than by anti-hFc gamma RIIa mAb. Inhibition studies with mAb against CR3 (B2.12) and hFc gamma RIIa showed that both receptors cooperated in phagocytosis. We conclude that PMN phagocytosis can be effectively mediated by antibacterial IgG2 anti-STAW and anti-Hib. Thus, IgG2 may have a critical role in the immune defense against these bacteria. However, the role of antibacterial IgG2 in opsonization and phagocytosis may be limited in individuals homozygous for hFc gamma RIIaHR.
Neuroprotective role of IL-4 against activated microglia.
Authors: Chao C C , Molitor T W , Hu S
Published: December 2022

Microglia have been proposed to play a pathogenetic role in immunologically mediated neurodegenerative diseases. In our study, using microglial/neuronal cell cocultures primed with IFN-gamma, we found that both LPS and TNF-alpha triggered neuronal cell injury (impairment of gamma-aminobutyric acid uptake and neuronal loss) via a nitric oxide mechanism. Pretreatment of cell cocultures with IL-4, an immunosuppressive cytokine, prevented, in a dose-dependent manner, neuronal cell injury induced by activated microglia. The mechanism by which IL-4 exerts its neuroprotective effect was found to involve the inhibition of IFN-gamma priming of microglia with a subsequent decrease in the production of TNF-alpha and nitric oxide.
Gene targeting yields a CD18-mutant mouse for study of inflammation.
Authors: Wilson R W , Ballantyne C M , Smith C W , Montgomery C , Bradley A , O'Brien W E , Beaudet A L
Published: December 2022

CD18 is the common beta subunit for the heterodimeric leukocyte integrins that mediate many inflammatory cell adhesion responses including binding to intercellular adhesion molecules 1 and 2. CD18 deficiency in humans causes a severe granulocyte disorder with susceptibility to bacterial infections and high morbidity and mortality. Gene targeting was used to prepare an insertion mutation in the murine CD18 gene. The insertion mutation resulted in a hypomorphic rather than a null allele due to low expression from a cryptic promoter in the plasmid construct. Homozygous mutant mice are viable and fertile, demonstrate a mild granulocytosis, and have 2 or 16% of normal CD18 expression on granulocytes in the resting or activated state, respectively. Mutant mice show an impaired inflammatory response to a chemical peritonitis and delayed rejection of cardiac transplants. These CD18-mutant mice provide a model of the moderate form of the human disease and should be extremely valuable for in vivo analysis of the role of leukocyte integrin-dependent adhesion in inflammatory disease models.
Relation between skin cancer, humoral responses to human papillomaviruses, and HLA class II molecules in renal transplant recipients.
Authors: Bavinck J N , Gissmann L , Claas F H , Van der Woude F J , Persijn G G , Ter Schegget J , Vermeer B J , Jochmus I , Müller M , Steger G
Published: December 2022

Human papillomaviruses (HPV), especially the epidermodysplasia verruciformis (EV)-associated HPV 5, 8, 14, 17, 20, and 47, are thought to play a role in the pathogenesis of some skin cancers in recipients of renal allografts. MHC class I and class II genes are involved in the cellular immune response to viral and tumor Ag. Little is known about humoral responses to HPV in recipients with and without skin cancer. We investigated the prevalence of antibodies to the early (E) protein E7 and the major capsid late (L) protein L1 of HPV 8. In addition, we studied the association of HLA class II molecules with these antibody responses. The E7 and L1 open reading frames of HPV 8 were bacterially expressed as beta-galactosidase fusion proteins, which were purified by preparative gel electrophoresis. Serum samples from 36 renal transplant recipients with and 91 recipients without skin cancer were screened for the presence of IgG and IgM antibodies to HPV 8 E7 and L1, by Western blot analysis. The detection of anti-HPV 8 L1 antibodies represents the immune response to HPV 8 and possibly other EV-associated HPV, because cross-reactivity between the representatives of this HPV subgenus can occur. The antibody responses to HLA Ag were used as controls. Recipients who had IgM antibodies but no IgG antibodies to L1 of HPV 8 (patients with no apparent class switch from IgM to IgG) had skin cancer in 50% of cases, whereas recipients who produced IgG antibodies (patients with an apparently good humoral response to L1 of HPV 8) had skin cancer in only 18% of cases. The estimated relative risk of skin cancer in recipients with no class switch, compared with the risk in those with a good humoral response, was 4.5 (95% confidence interval, 1.1 to 18.1). We found no association between the antibody response to HLA Ag and the occurrence of skin cancer. A strong linkage between the absent class switch of antibody production in response to L1 of HPV 8 and HLA-DR7 was observed (relative risk, 26.2). Renal transplant recipients who have no apparent class switch from IgM to IgG production in response to Ag encoded by L1 of HPV 8 or possibly other EV-associated HPV are at an increased risk of skin cancer. The association with HLA-DR7 indicates a genetic control of skin cancer development or regression, involving genes in the class II region of the MHC.