bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligation of CD23 triggers cAMP generation and release of inflammatory mediators in human monocytes.
Authors: Paul-Eugene N , Kolb J P , Abadie A , Gordon J , Delespesse G , Sarfati M , Mencia-Huerta J M , Braquet P , Dugas B
Published: December 2022

Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in normal human monocytes using monoclonal antibodies to CD23 (MHM6 and 135) and IgE/anti-IgE immune complexes. Monocytes expressing an increased amount of CD23 molecules were obtained by stimulation with IL-4 (30 U/ml). Anti-CD23 mAb as well as IgE/anti-IgE immune complexes were unable to induce any significant calcium mobilization [Ca2+]i in CD23-bearing monocytes whereas they elicited [Ca2+]i increase in B lymphocytes of the same donors. Despite their failure to induce calcium mobilization, the same CD23 ligands triggered a dose-dependent increase of intracellular cAMP, with a maximum 20 to 30 min after the onset of stimulation. This effect is mediated via CD23 inasmuch as: 1) F(ab)'2 fragments are as active as intact anti-CD23 mAb and 2) it is not observed in CD23- monocytes. The increase in cAMP was only partially altered in the presence of 1 microM indomethacin suggesting that it was not due to the release of PG. The possible role of CD23 in the activation of human monocytes was next documented by showing that anti-CD23 mAb and IgE/anti-IgE immune complexes induced the generation of IL-6 and of thromboxane B2 by CD23+ but not by CD23- monocytes. In addition, the IgE/anti-IgE-induced IL-6 production was potentiated in the presence of cAMP inducer such as the beta 2-adrenoceptor agonist salbutamol. These results indicate that ligation of CD23 induces cAMP generation in CD23+ human monocytes and that CD23 may regulate the IgE-dependent functions in normal human monocytes.
Selective decrease of CD26 expression in T cells from HIV-1-infected individuals.
Authors: Blazquez M V , Madueño J A , Gonzalez R , Jurado R , Bachovchin W W , Peña J , Muñoz E
Published: December 2022

The decrease of CD4+ cells in AIDS patients is widely documented, although the selective loss within different subsets of CD4+ cells and the mechanisms involved in this phenomenon are controversial. In the present report we have analyzed the proliferative response to Ag and mitogen of peripheral blood T lymphocytes from HIV-infected individuals, the phenotype profile of CD26+ and CD26- subset of cells and their infectivity by the HIV. The expression of CD26 Ag, either in CD4+ or CD8+ cells, was clearly diminished in all the patients tested. On the other hand, the expression of CD29 seems not to be affected, nevertheless T cells from these patients were unable to generate a proliferative response against soluble Ag. In 11 out of 13 patients, polymerase chain reaction studies demonstrated that the CD26- subset of CD4+ cells was the main reservoir for HIV-1 in infected individuals and HIV-1 virus preferentially infected in vitro CD4+/CD26- subpopulation. This capacity for preferential infectivity, together with the selective loss of cells expressing CD26 Ag, helps to explain the progressive impairment in the immune system of these patients and sheds new light on our understanding of the AIDS pathophysiology.
Heterogeneity in the recognition of the simian immunodeficiency virus envelope glycoprotein by CD4+ T cell clones from immunized macaques.
Authors: Jones W C , Barnard A L , Slade A , Mills K H
Published: December 2022

CD4+ T cell recognition of the simian immunodeficiency virus (SIV) surface envelope (env) glycoprotein was examined by using a panel of 10 T cell lines and 4 T cell clones derived from 10 individual macaques immunized with inactivated SIV or recombinant SIV env proteins. The results demonstrated that CD4+ T cells from each animal recognized between 1 and 7 peptides in 4 distinct regions of the protein including both variable and conserved domains. MLR of PBMC from selected macaques together with RFLP analysis by using the HLA DR beta probes suggested that animals of distinct MHC class II haplotypes can recognize identical peptides. These T cell epitopes within conserved regions of the envelope protein, together with identified linear B cell epitopes recognized by neutralizing antibodies, may be relevant in vaccine design.
Passive Transfer of the Skin Reaction to Horse Serum
Authors: Clarke J. A. , Gallagher May G.
Published: December 2022

The great advances made in the last few years in the treatment of disease by the use of anti-sera are marred by a few deaths from hypersensitiveness to horse serum. Individuals differ widely in their reactions to serum injections, so widely in fact, that thus far it is impossible to say how large a dose will be necessary to sensitize. Even the small dose administered in the diphtheria toxin-antoxin mixtures may result in, at least, skin sensitivity (1).

At the present time, the intradermal test is the most common method used to determine sensitivity in the person who is to receive the serum injection. A small amount of horse serum is injected between the layers of the skin, and if the skin is sensitive, an urticarial wheal will develop at the site of the injection.
IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity.
Authors: Sykes M , Abraham V S , Harty M W , Pearson D A
Published: December 2022

We have recently demonstrated, in a fully MHC-mismatched murine bone marrow transplantation model, that administration of a short course of high dose IL-2 markedly diminishes graft-vs-host disease (GVHD) without compromising alloengraftment or the graft-vs-leukemia (GVL) effect of allogeneic T cells. We have now evaluated the mechanism of the dissociation of GVL and GVHD observed in this model. We demonstrate that CD4+ T cells were required to produce severe, acute GVHD in the fully MHC-mismatched plus minor histocompatibility Ag-mismatched A/J-->B10 strain combination. The GVHD-producing activity of A/J CD4+ T cells administered without CD8+ T cells was inhibited by IL-2 treatment. In contrast, CD8+ T cells alone mediated the GVL effect observed in the EL4 leukemia/lymphoma model, and CD4+ cells did not contribute to this effect. This CD8-mediated GVL activity was not inhibited by IL-2 treatment. Because naive A/J CD8+ T cells administered without CD4+ T cells did not produce acute GVHD, we were unable to evaluate the effect of IL-2 in this model. However, when A/J donors were presensitized with B10 skin grafts, CD4-depleted A/J spleen cells were capable of causing acute GVHD in B10 recipients. This CD8-mediated GVHD was not inhibited by treatment with IL-2. However, IL-2 did partially inhibit the GVHD produced by nondepleted presensitized A/J spleen cells, probably due to selective inhibition of the function of presensitized A/J CD4+ T cells. The dissociation of GVHD and GVL against the EL4 leukemia/lymphoma in IL-2-treated mice can therefore be explained by selective inhibition by IL-2 of CD4 activity.
Receptor-mediated antigen delivery into macrophages. Complexing antigen to alpha 2-macroglobulin enhances presentation to T cells.
Authors: Chu C T , Pizzo S V
Published: December 2022

Macrophages secrete alpha 2-macroglobulin (alpha 2M), a protein that may facilitate early Ag handling. alpha 2M is able to entrap and form covalent linkages with diverse proteins during a transient proteinase-activated state. The resulting complexes are rapidly endocytosed after binding to high affinity receptors. Such a system could be capable of efficiently delivering a multitude of proteins to macrophages. We have used T hybridoma clones that respond only to hen egg lysozyme, in a MHC-restricted manner, to probe the effect of complex formation on Ag uptake and processing by murine macrophages. Radiolabeled lysozyme was internalized more rapidly and to a greater extent when bound to alpha 2M than when unbound. Macrophages pulsed with lysozyme-alpha 2M-elastase complexes required 200 to 250 times less Ag than those pulsed with free lysozyme to achieve effective presentation to T cells. Adding equimolar amounts of alpha 2M-elastase complexes, or of alpha 2M-methylamine, to free lysozyme had no effect on basal lysozyme presentation. Receptor-recognized forms of alpha 2M, but not lysozyme or BSA, competed effectively for both uptake and presentation of lysozyme-alpha 2M-elastase complexes. These results indicate that proteinase-activated alpha 2M can enhance Ag processing by carrying Ag into macrophages through a receptor-mediated process.
Production of DNP-specific/class I MHC-restricted suppressor molecules is linked to the expression of T cell receptor alpha- and beta-chain genes.
Authors: Fairchild R L , Palmer E , Moorhead J W
Published: December 2022

The hapten/class I MHC-specific soluble immunoregulatory molecules produced by CD8+T cells from dinitrobenzene sulfonate-primed mice express the binding specificity and serologic determinants of alpha/beta TCR. To examine the genes used to encode these soluble immunoregulatory molecules, we utilized a surface TCR expressing Ts hybridoma, which constitutively produces a DNP/Kd-specific regulatory molecule. Northern and Southern analyses indicated that MTs 79.1 cells use a V beta 8 and a V alpha 4 gene to encode the variable regions of the surface alpha/beta TCR. A panel of TCR- variants was generated by subjecting MTs 79.1 cells to gamma-irradiation. Twelve of the TCR- variants were chosen for detailed characterization. Northern blot analyses indicated the absence of the MTs 79.1 V alpha 4 chain transcript in five of the variants and the absence of the parental V beta 8 chain transcript in the other seven. Southern blot analyses demonstrated the deletion of the parental gene encoding the alpha- or beta-chain from the genome of the respective mutant. None of the 12 TCR gene deletion mutants produced the parental suppressive activity. Expression of the parental TCR beta-chain gene in one of the beta-chain gene deletion mutants reconstituted the ability to produce this activity. As with the MTs 79.1 molecule, the regulatory molecule produced by the beta-chain gene transfectant was bound by and eluted from Sepharose columns coupled with either DNP or anti-V beta 8 antibodies. These results establish a strong linkage between the suppressor molecules produced by these Ts and TCR alpha- and beta-chain gene transcription.
MHC class II limits the functional expression of endogenous superantigens in B cells.
Authors: Lund F E , Randall T D , Woodland D L , Corley R B
Published: December 2022

The open reading frames of the 3' long terminal repeat of mouse mammary tumor viruses (MMTV) encode superantigens. When expressed with MHC class II molecules on the surface of B cells, superantigens stimulate T cells expressing receptors with particular V beta elements. By using B cell lymphomas as model systems, we demonstrate that stimuli, including certain lymphokines (IL-2 and IL-5) and LPS, that lead to increased MMTV transcript levels in B cells did not increase functional superantigen presentation. Furthermore, stimulation of BCL1 and CH12 cells with LPS resulted in a pronounced reduction in superantigen presentation, even though MMTV transcript levels were increased. In contrast to these effects, functional superantigen expression was increased under conditions in which class II levels were increased, even when MMTV levels remained constant, indicating that the levels of newly synthesized class II are important for functional superantigen expression. These data together imply that the amount of cell surface class II limits superantigen presentation and/or that superantigens can associate only with a limited pool of class II.
Dissociation of antigen-presenting capacity of astrocytes for peptide-antigens versus superantigens.
Authors: Rott O , Tontsch U , Fleischer B
Published: December 2022

Although superantigens and their molecular interactions with MHC class II molecules have been well characterized recently, little is known concerning the physiological function of different types of APC in inducing superantigen-mediated T cell activation. To evaluate the potential of nonhematopoetic cells to present superantigens to T cells, we have tested astrocytes as a typical "nonprofessional" APC. Although astrocytes can express appropriate levels of MHC class II products and adhesion molecules, they turned out to be unable to mediate superantigen-driven activation of normal T lymphocytes, even in the presence of rather high concentrations of toxins. In contrast, they could properly present equimolar amounts of nominal Ag to various Ag-specific T cell lines under the same experimental conditions. Inability of astrocytes to support T cell responses to superantigens could not be overcome by addition of cytokines IL-1 and IL-6. Binding studies with class II-expressing astrocytes revealed that T cell unresponsiveness was not due to a general failure of astrocytes to bind the superantigen. Moreover, the resulting SA-class II complex was recognizable by TCR, as demonstrated by the capacity to activate IL-2 secretion in T cell hybridomas. Our results extend previous studies demonstrating marked differences of various types of APC to trigger T cell responses to superantigens and describe for the first time a dissociation of the Ag-presenting capacity for peptide-Ag vs superantigen on an accessory cell.
Insulin regulates serine/threonine phosphorylation in activated human B lymphocytes.
Authors: Valentine M A , Licciardi K A , Clark E A , Krebs E G , Meier K E
Published: December 2022

Activation of either dense tonsilar B lymphocytes or the B lymphoblastoid cell line T5-1 with Staphylococcus aureus, Cowan strain I, induced surface expression of insulin receptors. Addition of insulin to these activated cells resulted in subsequent phosphorylation of the B cell surface protein CD20, the functions to regulate B cell activation. The cytoplasmic domains of CD20 contain multiple serine and threonine residues, of which at least two are followed by acidic sequences typical of substrate sites favored by casein kinase II. Tryptic mapping of CD20 isolated from intact cells treated with insulin showed increased phosphorylation on peptides having similar migration to those phosphorylated by casein kinase II in vitro. Treatment of tonsilar B cells or T5-1 cells with phorbol esters or in vitro phosphorylation by purified protein kinase C did not result in phosphorylation of peptides phosphorylated by casein kinase II, suggesting that protein kinase C is not directly involved in CD20 phosphorylation in the response to insulin. Phosphorylation of CD20 cannot be triggered by insulin in resting B cells, as the insulin receptor is expressed only after entry into the G1 phase of the cell cycle. Thus, distinct regulation of activation pathways are available to resting as opposed to activated B lymphocytes.
Expression of polyamine transporter activity during B lymphocyte cell cycle progression.
Authors: DeBenedette M , Olson J W , Snow E C
Published: December 2022

The movement of resting B cells into the early stages of the cell cycle is accompanied by elevations in cellular polyamine levels. Our previous results indicate that part of this increase is a consequence of enhanced levels of ornithine decarboxylase activity in activated B cells. However, the levels of ornithine decarboxylase wane within 30 h post-stimulation, suggesting that B cells may acquire polyamines from an alternative source. Results in the present communication indicate that B cells initiate the transport of extracellular polyamines as they enter the G1 stage of the cell cycle. This induction of polyamine transport activity is seen after the stimulation of B cell cycle progression by both PMA and ionomycin, as well as cognate help from activated Th cells. The transport activities are energy and temperature sensitive, and can be competitively inhibited by unlabeled ligands. The results of the competition studies are compatible with the presence of at least two types of transporters, a low affinity transporter for both putrescine and spermidine, and a higher affinity transporter selective for spermidine. The results are discussed in terms of biochemical events associated with Th cell-mediated, B cell activation.
Antibodies to CD44 trigger effector functions of human T cell clones.
Authors: Galandrini R , Albi N , Tripodi G , Zarcone D , Terenzi A , Moretta A , Grossi C E , Velardi A
Published: December 2022

mAb against the lymphocyte homing receptor CD44/Hermes up-regulate the proliferation of human T PBL induced by anti-CD3 or anti-CD2 mAb. Moreover, certain anti-CD44 mAb can activate human resting T cells and mouse cytotoxic T cells in the absence of anti-CD3 or anti-CD2 mAb. Here, we show that anti-CD44 mAb trigger proliferation of human CD3+/CD4+ T cell clones in a fashion similar to that observed with mAb to CD3. Such an effect is IL-2-dependent, as shown by IL-2 production induced by anti-CD44 mAb and by complete inhibition of cell proliferation in the presence of anti-IL-2 antibodies or cyclosporin A. Moreover, anti-CD44 mAb trigger human cytolytic T cell clones to lyse Fc gamma-R+ P815 cells in the absence of additional stimuli. The magnitude of the cytolytic response induced by anti-CD44 mAb is comparable to that observed in the presence of anti-CD3 mAb for both CD4+ and CD8+ TCR-alpha/beta+ clones, and for V delta 1 or V delta 2 TCR-gamma/delta+ clones. By contrast, in CD3-/CD16+ NK cell clones, no cytolytic responses to anti-CD44 mAb could be observed. Granule trypsin-like esterase enzyme (granzyme) release by cytolytic T cell clones is induced by plastic-immobilized anti-CD44 mAb. Anti-CD44 mAb-triggered proliferation ([3H]thymidine incorporation) and cytotoxicity are blocked by the protein tyrosine kinase inhibitor, genestein. In addition, ligation of the CD44 molecule induces tyrosine phosphorylation of proteins identical, by molecular mass, to those phosphorylated after anti-CD3 mAb stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21-kDa protein (the phosphorylated zeta-chain of the TCR molecular complex) typically observed upon anti-CD3 mAb stimulation. In conclusion, this study shows that the ligated CD44 molecule provides the necessary stimuli for a variety of T cell-mediated functions triggered via protein tyrosine kinase-dependent signal transduction pathways at least in part similar to those that follow stimulation of the CD3/TCR complex.
Superantigen-induced peripheral tolerance inhibits T cell responses to immunogenic peptides in TCR (beta-chain) transgenic mice.
Authors: Perkins D L , Wang Y , Ho S S , Wiens G R , Seidman J G , Rimm I J
Published: December 2022

TCR (beta-chain) transgenic mice were tolerized with the superantigen staphylococcal enterotoxin B (SEB). Three to 28 days after tolerization with SEB, flow cytometry of peripheral T cells showed the persistence of SEB-unresponsive T cells that did not express reduced levels of the TCR (beta-chain) transgene. Stimulation of the tolerized T cells with a panel of superantigens (SEC1), mitogens (Con A, PHA, and pertussis toxin) and mAb (anti-CD3 epsilon) did not induce T cell proliferation. In contrast to other models, exogenous rIL-2 did not reverse unresponsiveness and induce proliferation. In addition, lymphokines rIL-4 and rIL-6 also did not induce proliferation. However, the unresponsive T cells did respond to the combination of PMA plus ionomycin, but not to PMA or ionomycin alone. Thus, the block in signal transduction in the anergic state occurs between the stimulation of cell surface receptors and the activation of protein kinase C and the increase in intracellular calcium. In addition, these results show that mature T cells tolerized with the superantigen SEB are unresponsive to a wide array of T cell stimuli, indicating a block in a common signal transduction pathway.
Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells.
Authors: Wesselborg S , Janssen O , Kabelitz D
Published: December 2022

Signaling via the CD3/TCR complex induces programmed cell death (apoptosis) in immature thymocytes and transformed T lymphocytes (hybridomas or leukemic cells). Accumulating evidence indicates, however, that apoptosis can be triggered also in mature peripheral T cells. Here we show that a significant fraction of cells of a given IL-2-dependent TCR-alpha beta + clone or polyclonal short term line is killed when cultured for 20 h in the presence of PHA, anti-CD3 (OKT3), or anti-TCR (BMA031) mAb. Apoptosis can be triggered by these stimuli in CD4+, CD8+, and CD4-CD8- (double-negative) TCR-alpha beta+ clones. Activation-driven cell death (as quantified by propidium iodide staining and FACS analysis) is associated with fragmentation of DNA into oligonucleosomal bands of approximately 200 bp. Although freshly isolated peripheral blood T cells are largely resistant to apoptosis, the sensitivity to anti-CD3/TCR mAb or PHA-triggered cell death gradually increases upon activation and IL-2-dependent culture of T cells, and reaches a plateau level after 15 to 20 days. These data indicate that stimuli that activate resting T cells initiate death by apoptosis in activated T cells. The implications of these results for the regulation of cellular immune responses and the establishment of peripheral tolerance will be discussed.
Regulated expression of germline antigen receptor genes in mast cell lines from the murine embryo.
Authors: Siden E J
Published: December 2022

Abelson murine leukemia virus-transformed cell lines derived from the earliest period of murine embryonic hematopoiesis express multiple characteristics of immature mast cells. We show here that both Ig and TCR-gamma genes are transcribed in some of these embryo-derived mast cell lines. Germline H chain V region transcripts are expressed constitutively, and germline Ig-mu and TCR-gamma constant region gene transcripts are induced in culture by the antiproliferative drug, BUdR. Coordinate with the up-regulation of the receptor gene transcripts, the B cell surface protein, B220, and IL-4 mRNA are also induced. The mechanism of action of BUdR was revealed by the observation that exogenous IL-4 alone induced both mu- and TCR transcripts in the transformed cells. Nontransformed mast cells cultured from embryonic liver and placenta also contain mu- and TCR-gamma transcripts. The expression of multiple Ag receptor genes in mast cells suggests that this cell type may be useful for our understanding of some of the early events of lymphoid development.
Nonimmunogenic tumor cells may efficiently restimulate tumor antigen-specific cytotoxic T cells.
Authors: Kündig T M , Bachmann M F , Lefrancois L , Puddington L , Hengartner H , Zinkernagel R M
Published: December 2022

Induction of immunity to a viral protein that had been transfected into a tumor cell line was studied. The nucleoprotein (NP) of vesicular stomatitis virus (VSV) was used as a model tumor-associated Ag after transfection into EL-4, and H-2b thymoma originating from C57BL/6 mice. The NP-transfected cell line (EL-4NP) was lysed by NP-specific CTL and was found to restimulate NP-specific CTL in vitro as efficiently as did VSV-infected macrophages. Despite both of these in vitro characteristics, C57BL/6 mice inoculated with EL-4NP did not mount a measurable NP-specific CTL response and developed a lethal tumor as rapidly as did mice given control EL-4. This lack of immunogenicity could not be explained by down-regulation of MHC class I molecules or by loss of NP; even EL-4NP cells metastasizing to the spleen kept their high restimulatory capacity and excellent target characteristics. However, once mice were immunized with VSV or with a vaccinia-VSV-NP recombinant virus they were protected against tumor growth of EL-4NP by CD8+ CTL but not by CD4+ T cells. Taken together, the failure of the tumor-associated Ag to induce a protective T cell response in vivo despite its excellent capacity to restimulate CTL in vitro may encourage adjuvant immunotherapy in cancer; even the effects of weakly immunizing tumor vaccines, e.g., recombinant viruses, may be efficiently amplified by tumor cells.
Blocking lymphoma invasiveness with a monoclonal antibody directed against the beta-chain of the leukocyte adhesion molecule (CD18).
Authors: Zahalka M A , Okon E , Naor D
Published: December 2022

The same integrin adhesion molecules used by normal leukocytes for traffic and localization in inflammation sites may be used by malignant cells for dissemination. Identifying the adhesion molecules and then blocking them with appropriate antibody may therefore prove useful for controlling tumor spread. This prediction was tested on a spontaneous murine T cell lymphoma (LB) that expresses LFA-1 adhesion molecules. The adhesion molecules were identified by fluorocytometry and immunoprecipitation with anti-CD18 mAb (M18/2). Subcutaneously inoculated LB lymphoma rapidly infiltrated the spleen and the lymph nodes, as indicated by histologic examination and [3H]thymidine incorporation assay of proliferating LB cells derived from the invaded organs. The normal organization of the lymphoid organs was totally effaced by the infiltrating LB cells. Intravenous injection of anti-CD18 mAb, protein G-purified anti-CD18 mAb, or its F(ab')2 fragments (but not irrelevant control mAb) blocked the invasion of the s.c. inoculated lymphoma into the spleen. Whereas i.v. injected anti-CD18 mAb could not block the infiltration of LB cells into the lymph nodes, local s.c. injection of this antibody near the lymph nodes partially inhibited lymphoma invasion into these organs. It was further found that LB cells form aggregates with spleen cells but not with lymph node cells. In addition, spleen-infiltrating LB cells invade both the spleen and the lymph nodes after s.c. injection. On the other hand, lymph node-infiltrating LB cells invade mainly the lymph nodes under similar circumstances.
MCP-1 expression by rat type II alveolar epithelial cells in primary culture.
Authors: Paine R , Rolfe M W , Standiford T J , Burdick M D , Rollins B J , Strieter R M
Published: December 2022

Recruitment and activation of mononuclear phagocytes are potentially critical regulatory events for control of pulmonary inflammation. Located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells are ideally situated to regulate the recruitment and activation of mononuclear phagocytes through the production of cytokines in response to inflammatory stimulation from the alveolar space. To test this hypothesis, we investigated the production of monocyte chemotactic polypeptide-1 (MCP-1), a protein that is chemotactic for and that activates monocytes, by rat type II alveolar epithelial cells in primary culture. Immunocytochemical staining using anti-murine JE, an antibody recognizing rat MCP-1, demonstrated cell-associated MCP-1 Ag throughout the monolayer. The intensity of staining was increased in response to IL-1 beta. When type II epithelial cells formed a tight monolayer on a filter support, there was polar secretion of MCP-1 Ag into the apical compartment by both control and IL-1-stimulated cells as measured by specific MCP-1 ELISA. Northern blot analysis revealed that IL-1 and TNF-alpha stimulated MCP-1 mRNA expression in a dose-dependent manner, whereas dexamethasone blocked MCP-1 expression by cells stimulated with IL-1. In contrast to previous results using transformed epithelial cell lines, MCP-1 mRNA was induced in these primary cultures directly by stimulation with LPS. These data suggest that alveolar epithelial cells may have an important and previously unrecognized role in the initiation and maintenance of inflammatory processes in the lung by recruiting and activating circulating monocytes through the production of MCP-1.
Cyclosporin H is a potent and selective formyl peptide receptor antagonist. Comparison with N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L- leucyl-L-phenylalanine and cyclosporins A, B, C, D, and E.
Authors: Wenzel-Seifert K , Seifert R
Published: December 2022

The cyclic undecapeptide, cyclosporin (Cs) H, is a potent inhibitor of FMLP-induced superoxide anion (O2-) formation in human neutrophils. We studied the effects of CsH in comparison with those of N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine (BocPLPLP), a well known formyl peptide receptor antagonist, and of other Cs on activation of N6,2'-O-dibutyryl adenosine 3:5'-monophosphate-differentiated HL-60 cells and human erythroleukemia cells (HEL cells). CsH inhibited FMLP binding in HL-60 membranes with a Ki (inhibition constant) of 0.10 microM. CsH inhibited activation by FMLP of high affinity GTPase (the enzymatic activity of alpha-subunits of heterotrimeric regulatory guanine nucleotide-binding proteins) in HL-60 membranes with a Ki of 0.79 microM. CsH inhibited the stimulatory effects of FMLP on cytosolic Ca2+ concentration ([Ca2+]i), O2- formation, and beta-glucuronidase release with Ki values of 0.08, 0.24, and 0.45 microM, respectively. BocPLPLP was 14-fold less potent than CsH in inhibiting FMLP binding and 4- to 6-fold less potent than CsH in inhibiting FMLP-induced GTP hydrolysis, rises in [Ca2+]i, O2- formation, and beta-glucuronidase release. CsA reduced FMLP-induced O2- formation by 20%, but CsB, CsC, CsD, and CsE did not. CsA, CsB, CsC, CsD, and CsE did not affect FMLP-induced rises in [Ca2+]i. BocPLPLP inhibited leukotriene B4-induced rises in [Ca2+]i with a Ki of 0.33 microM, whereas CsH showed no inhibitory effect. CsH and BocPLPLP did not inhibit the rises in [Ca2+]i induced by several other stimuli in HL-60 cells and HEL cells. Our results show that 1) CsH is a more potent formyl peptide receptor antagonist than BocPLPLP; 2) unlike BocPLPLP, CsH is selective; and 3) N-methyl-D-valine which is present at position 11 of the amino acid sequence of CsH but not of other Cs is crucial for FMLP antagonism.
Potent stimulation of monocytic endothelin-1 production by HIV-1 glycoprotein 120.
Authors: Ehrenreich H , Rieckmann P , Sinowatz F , Weih K A , Arthur L O , Goebel F D , Burd P R , Coligan J E , Clouse K A
Published: December 2022

Monocytes/macrophages play a critical role in the pathogenesis of HIV infection, both as targets for virus replication and as sources of production of multifunctional cytokines. Endothelins, peptides with potent vasoconstricting activities originally isolated from endothelial cells, are also produced and secreted by macrophages in a manner similar to that of other cytokines. In an attempt to explore the potential role of endothelins in HIV-infection, we investigated the effect of the HIV-1 envelope glycoprotein, glycoprotein 120, on monocytic endothelin-1 production. This glycoprotein has been identified as a potent stimulator of monokines such as TNF-alpha and IL-6, which have been implicated as potential mediators of HIV-encephalopathy. We found that glycoprotein 120, similar to LPS, stimulates the secretion of endothelin-1, as well as TNF-alpha, from macrophages in a concentration-dependent manner. Using reverse transcriptase polymerase chain reaction, we found that circulating monocytes in HIV-infected individuals show a distinct expression of the endothelin-1 gene that is not detectable in healthy controls, indicating chronic activation of this gene in HIV-infection. In addition, cerebral macrophages in patients with HIV-encephalopathy were strongly positive for endothelin. Thus, monocytic endothelins appear to be stimulated during HIV infection. Their potent vasoactive properties render them potential candidates for mediating alterations in the cerebral perfusion pattern associated with the AIDS dementia complex.
Induction of circulating IL-1 receptor antagonist by IFN treatment.
Authors: Tilg H , Mier J W , Vogel W , Aulitzky W E , Wiedermann C J , Vannier E , Huber C , Dinarello C A
Published: December 2022

This study was undertaken to determine whether IFN induce IL-1 receptor antagonist (IL-1Ra), a specific inhibitor of IL-1. Plasma samples were obtained from healthy volunteers (n = 5) and patients with chronic hepatitis C (n = 5) treated with IFN-alpha, and from patients with renal cell carcinoma (n = 6) treated with IFN-gamma and assayed for IL-1Ra by a specific radioimmunoassay. Both types of IFN were administered subcutaneously. In vitro studies were carried out with PBMC from healthy volunteers. A single, low and nontoxic dose (1 x 10(6) U) of IFN-alpha induced circulating IL-1Ra, which reached peak levels within 12 h. This effect was dose-dependent and more pronounced with a higher dose (5 x 10(6) U). Peak IL-1Ra levels 12 h after 5 x 10(6) U IFN-alpha were 4.16 +/- 0.35 ng/ml in healthy volunteers and 5.7 +/- 0.73 ng/ml in patients with chronic hepatitis C (difference not significant). Thereafter levels declined but remained elevated for 24 h. IFN-gamma treatment led only to a modest increase of circulating IL-1Ra even at a dose of 400 micrograms; this dose, however, was associated with side effects similar to those seen after injection of 5 x 10(6) U IFN-alpha. PBMC stimulated with IFN-alpha or IFN-gamma produced IL-1Ra in vitro. The induction of IL-1Ra may contribute to the antiviral, anti-inflammatory, and antiproliferative effects of IFN.
Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10.
Authors: Daeipour M , Kumar G , Amaral M C , Nel A E
Published: December 2022

IL-6 is a multi-functional cytokine that utilizes 80-kDa ligand-binding and 130-kDa signal-transducing subunits to stimulate diverse cellular responses. Although IL-6R ligation has been associated with tyrosine protein phosphorylation and activation of an unidentified serine/threonine kinase, very little is known about the intermediary signaling events between the cell membrane and the nucleus. rIL-6 treatment of the human B cell line, AF-10, induced MAP kinase (mitogen-activated protein kinase) activity as determined by in vitro phosphorylation of microtubule-associated protein-2 (MAP-2) and the synthetic peptide APRTPGGRR, corresponding to amino acids 95-98 of bovine myelin basic protein. The kinetics of the response was rapid and dependent on the dose of rIL-6. The response was cytokine specific, did not require the presence of extracellular Ca2+, and was minimally affected by the presence of staurosporine. MAP kinase activation in AF-10 cells occurred in parallel with appearance of 42- and 44-kDa tyrosine phosphoproteins (p42 and p44). Moreover, MAP kinase activation was diminished when AF-10 cells were stimulated with rIL-6 in the presence of tyrosine protein kinase inhibitors, genistein and geldanomycin. p42 and p44 co-electrophoresed on SDS-PAGE with extracellular signal-related kinase (ERK)-2, and ERK-1, respectively; both are members of the ERK family. In addition to p42MAPK and p44MAPK, rIL-6 also activated a MAP-2 kinase that eluted at a lower salt concentration (20 to 60 mM NaCl, peak I) from Mono-Q resin than p42MAPK (120 to 180 mM NaCl, peak II). The identify of this kinase is unknown but it is not an MPB kinase or a protein that exhibits immunoreactivity with anti-ERK antisera. In another IL-6-responsive B cell line, SKW6.4, rIL-6-activated peak I MAP-2 kinase but failed to activate ERK-2. The protein kinase C agonist, PMA, did, however, activate ERK-2 in SKW6.4 cells. These results show that the pleiotrophic cytokine, IL-6, activates p42MAPK/ERK-2 and at least one other serine/threonine kinase in B cell lines.
Mixed haplotype A beta Z/A alpha d class II molecule in (NZB x NZW)F1 mice detected by T cell clones.
Authors: Gotoh Y , Takashima H , Noguchi K , Nishimura H , Tokushima M , Shirai T , Kimoto M
Published: December 2022

We have tried to demonstrate the existence of a mixed haplotype MHC class II molecule in (NZB x NZW)F1 (B/WF1) mice. When a large panel of keyhole limpet hemocyanin-specific T cell clones derived from B/WF1 mice was analyzed, several clones were shown to be restricted by a F1-specific A beta Z/A alpha d class II molecule. Autoreactive A beta Z/A alpha d-specific T cell clones were also obtained. The ability of the association and expression of A beta Z with A alpha d was confirmed by hybridoma and transfection experiments. Hybridoma cell lines created by fusion of NZW (H-2z) spleen cells with M12.C3 (a A beta d- variant cell line derived from M12.4.1 (H-2d) B lymphoma) cells expressed A beta Z determinants. Transfection of A beta Z genomic DNA to M12.C3 cells resulted in the expression of A beta Z determinants. These hybridoma cell lines and transfectants were able to stimulate A beta Z/A alpha d-specific T cell clones, suggesting the expression of A beta Z/A alpha d molecules on the cell surface. However, attempts to demonstrate the existence of mixed haplotype MHC class II molecules in B/WF1 mice by two-dimensional (nonequilibrium pH gradient gel electrophoresis/SDS-PAGE) gel electrophoresis analysis with the use of anti-class II mAb failed to demonstrate the existence of mixed haplotype A beta Z/A alpha d or A beta d/A alpha z class II molecules in B/WF1 mice. Analysis of mixture of TA beta Z cell and B/WF1 spleen cell lysates immunoprecipitated by anti-A beta Z mAb suggested that the amount of haplotype mixed A beta Z/A alpha d molecules in B/WF1 spleen cells is less than 1/10 that of haplotype matched A beta/A alpha pairs. Our results suggest that, although undetectable by biochemical analysis, small amounts of mixed haplotype A beta Z/A alpha d molecules exist in B/WF1 spleen cells. Also, T cell clones which recognize them exists in B/WF1 mice. Because autoimmune symptoms of B/WF1 mice are shown to be related to heterozygosity at the H-2 region, autoreactive T cell clones which recognize the mixed haplotype A beta Z/A alpha d class II molecule might be involved for the induction of autoimmunity in B/WF1 mice.
Cholera toxin promotes B cell isotype switching by two different mechanisms. cAMP induction augments germ-line Ig H-chain RNA transcripts whereas membrane ganglioside GM1-receptor binding enhances later events in differentiation.
Authors: Lycke N Y
Published: December 2022

In a recent study we provided evidence that isotype switching in LPS-stimulated murine B cells was greatly enhanced by cholera toxin (CT). We found that CT acted synergistically with IL-4 to promote IgG1 differentiation at the gene transcriptional level by strongly enhancing the expression of germ-line gamma 1-RNA transcripts. In this study we ask which mechanisms are responsible for the isotype-switching effect of CT on B cells and which second messenger systems are involved in this process. We found that at least two different mechanisms are involved: 1) increased intracellular cAMP levels stimulated by the A subunit potentiates isotype switching early in differentiation by augmenting the formation of sterile germ-line gamma 1-RNA transcripts and 2) the binding of the nontoxic B subunit to the membrane GM1-ganglioside receptor promotes later stages of differentiation. Although the whole toxin gave up to a ninefold increase in IgG1 differentiation the cAMP-independent effect of rCTB gave at most a fivefold increase in IgG1 differentiation as compared to that seen with IL-4 alone. However, on a molar basis whole CT was at least a 1000-fold more efficient at promoting B cell switch-differentiation as compared to rCTB. Moreover, IL-4 did not stimulate cAMP in murine B cells and its effect on LPS-stimulated B cell differentiation was not decreased by inhibitors of cAMP-dependent protein kinases. However, CT's effect on B cell switch differentiation was blocked by inhibitors of protein kinases and could be partially mimicked by dibutyryl cAMP. In contrast to CT, the enhancing effect of rCTB on IgG1-differentiation was not affected by blocking of the protein kinases and the combination of rCTB and dBcAMP was as potent as the intact CT in promoting IL-4-stimulated IgG1 differentiation. Finally, the IL-4 pathway, but not the CT pathway, was sensitive to phorbol esters: In IL-4 plus LPS-stimulated B cell cultures IgG1 production was almost completely blocked by PMA. This inhibition was not associated with a decreased B cell proliferation or expression of germ-line gamma 1-RNA transcripts. The addition of CT, and to a significantly lesser extent rCTB, to these cultures enhanced IgG1-differentiation and expression of germ-line gamma 1-RNA transcripts to the same extent as in cultures without PMA. The existence of dual mechanisms operating together on B cell differentiation help to explain the strong adjuvant function by CT on IgG and IgA antibody responses after oral and parenteral immunizations.
Liver gamma delta T cells. TCR junctions reveal differences in heat shock protein-60-reactive cells in liver and spleen.
Authors: Roark C E , Vollmer M K , Cranfill R L , Carding S R , Born W K , O'Brien R L
Published: December 2022

The liver of mice contains elevated percentages of gamma delta T cells when compared with peripheral lymphoid organs. We have now analyzed these cells clonally, by generating a random collection of liver gamma delta T cell hybridomas and sequencing the productively rearranged TCR-gamma and -delta genes in each hybridoma clone. Examining C57BL/10 mice of various ages, we have found that over half of their normal gamma delta T cells are one of two types, V delta 4+ or V delta 6.3+. gamma delta T cell hybridomas generated from mouse liver contain clones that are "spontaneously" reactive, and respond to purified protein derivative from mycobacteria and to a 17-amino acid peptide from mycobacterial heat shock protein-60 (HSP-60). Like similar cells found in newborn thymus or adult spleen, all of the cells showing this HSP-60 reactivity pattern were found to express V gamma 1-J gamma 4-C gamma 4, most in conjunction with V delta 6-J delta 1-C delta, particularly with V delta 6.3. However, the gamma and delta junctional sequences of the V gamma 1/V delta 6+ cells isolated from adult liver differed from those found in adult spleen; being less diverse, their receptors instead resemble those of similar cells from newborn thymus. These data suggest that HSP-60-reactive gamma delta cells in adult murine liver and spleen are independent of each other and may be resident in their respective sites.
Rejection of an IA+ variant line of FBL-3 leukemia by cytotoxic T lymphocytes with CD4+ and CD4-CD8- T cell receptor-alpha beta phenotypes generated in CD8-depleted C57BL/6 mice.
Authors: Yoshimura A , Shiku H , Nakayama E
Published: December 2022

FBL-3N is an MHC class II Ag+ variant line that was obtained spontaneously during maintenance of Friend virus-induced leukemia FBL-3 in an athymic C57BL/6 (B6) mouse. Inocula of FBL-3N, but not the parental FBL-3 tumor, regressed after initial growth in CD8-depleted, syngeneic B6 mice. The cellular mechanisms by which FBL-3N was rejected in these mice were investigated in this study. We demonstrated that CTL with both CD4+ and CD4-CD8-TCR-alpha beta phenotypes were generated in mixed lymphocyte tumor cell culture spleen cells obtained from CD8-depleted B6 mice that had rejected FBL-3N by in vitro stimulation with mitomycin C-treated FBL-3N. After adoptive transfer of these CTL that were generated in vitro into athymic B6 mice, challenge with the FBL-3N tumor resulted in tumor regression after its initial growth. Thus, CD4+ and CD4-CD8-TCR-alpha beta CTL mediated rejection of the FBL-3N tumor in CD8-depleted B6 mice. Furthermore, the findings that depletion of B6 mice of CD4+ cells in addition to CD8+ cells abrogated the rejection of FBL-3N and generation of CTL in mixed lymphocyte tumor cell culture spleen cells suggest that CD4+ cells were required not only as a source of CD4+ CTL, but also as helper cells for generation of CD4-CD8-TCR-alpha beta CTL. Tumor Ag recognition of CD4-CD8-TCR-alpha beta CTL was restricted to Db, like that of classical CD8+ CTL, but the restriction appeared to be less obligatory than that of CD8+ CTL.
Residues that mediate DNA binding of autoimmune antibodies.
Authors: Radic M Z , Mackle J , Erikson J , Mol C , Anderson W F , Weigert M
Published: December 2022

Somatic mutations to arginine (R) are a common feature of a subset of J558 H chain genes that code for the majority of high-affinity, anti-dsDNA antibodies in autoimmune MRL/lpr mice. To examine the consequences of such amino acid substitutions on DNA binding, we reverted three somatic mutations of a prototypic anti-dsDNA H chain gene, VH3H9, and assayed the effect of those reversions by expression in a V lambda 1 L chain-only plasmacytoma line. Reversion of R53 eliminated virtually all dsDNA binding and sharply reduced ssDNA affinity. While the complete germ-line revertant of VH3H9 retained a low level of DNA binding, the substitution of R96, a product of N base addition in the third complementarity determining region (CDR3), with glycine (G) was sufficient to abolish measureable DNA specificity. Antibodies with higher affinity for DNA were generated by introducing arginines into VH3H9 at any one of four positions where somatic mutations to arginine had been identified by sequencing other anti-dsDNA J558 H chain genes. All four arginine mutants showed affinity increments consistent with their direct involvement in DNA binding, although one such mutant, K64R, required the simultaneous reversion of an adjacent aspartic acid (D) to the germ-line glycine. Two variants with three nongerm-line arginines showed further improvements in DNA affinity suggesting that their contributions to DNA binding may be additive. Molecular modeling of antibody and mutant F(ab) structures and calculations of their electrostatic potentials were used as an aid in interpreting the results and in predicting the location and size of possible combining sites.
Transfected Leishmania expressing biologically active IFN-gamma.
Authors: Tobin J F , Reiner S L , Hatam F , Zheng S , Leptak C L , Wirth D F , Locksley R M
Published: December 2022

Infection of susceptible BALB/c mice with Leishmania major leads to progressive infection with the failure to expand and activate Th1 CD4+ T cells that elaborate IFN-gamma, a critically implicated cytokine for control of disease. We used the recently described capacity to express foreign genes in trypanosomatids to introduce into Leishmania the murine IFN-gamma gene on a drug-selectable plasmid under the constitutive control of intergenic tubulin sequences. Several clones of L. major were established and demonstrated to contain IFN-gamma DNA and IFN-gamma RNA that was appropriately trans-spliced with the Leishmania-specific leader sequence, and to secrete IFN-gamma into the media. The secreted IFN-gamma was biologically active as assessed by up-regulation of class II MHC Ag and induction of macrophage nitric oxide synthase activity in a macrophage cell line. Infection of nude mice with IFN-gamma-containing organisms resulted in significantly slower progression of disease as compared to infection with organisms containing the empty plasmid, suggesting that biologically important activation of infected macrophages might be occurring in vivo. Infection of genetically susceptible BALB/c mice, however, did not impede the expansion of Th2 cells and the inexorable progression of disease. Despite the demonstration of increased levels of IFN-gamma transcription in vivo, induction of nitric oxide synthase in macrophages and expression of Ly-6, and IFN-gamma-inducible Ag, on CD4+ lymphocytes could not be shown. In all cases, organisms recovered from tissue amastigotes contained the IFN-gamma plasmid and secreted active IFN-gamma. The data confirm earlier studies that IFN-gamma alone is not sufficient to impede activation and maturation of Th2 cells in susceptible mice, even when targeted directly to the infected cell.