Showing papers by "National Jewish Health published in 1994"

Differential Activation of ERK and JNK Mitogen-Activated Protein Kinases by Raf-1 and MEKK

Abstract: Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.

Regulation of chemotaxis by the platelet-derived growth factor receptor-β

Abstract: Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.

Ras-Dependent Growth Factor Regulation of MEK Kinase in PC12 Cells

Abstract: Mitogen-activated protein kinases (MAPKs) are rapidly activated in response to stimulation of diverse receptor types. MAPKs are positively regulated by phosphorylation on threonine and tyrosine by MAP kinase or extracellular signal-regulated kinase (ERK) kinases (MEKs). MEK kinase (MEKK) is part of a family of serine-threonine protein kinases that phosphorylate and activate MEKs independently of Raf. MEKK was rapidly and persistently activated in response to stimulation of resting PC12 cells with epidermal growth factor (EGF). Nerve growth factor (NGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated MEKK, although to a lesser degree than did EGF. Activation of MEKK and B-Raf in response to EGF was inhibited by expression of dominant negative N17Ras. Expression of oncogenic Ras resulted in activation of MEKK. Stimulation of synthesis of cyclic adenosine 3',5'-monophosphate abolished activation of MEKK and B-Raf by EGF, NGF, and TPA. Thus, Ras simultaneously controls the activation of members of the Raf and MEKK families of protein kinases.

Immune protection and control of inflammatory tissue necrosis by gamma delta T cells.

Abstract: Host defenses against experimental listeriosis in mice involve neutrophils, macrophages, NK cells, and alpha beta T cells. Recently gamma delta T cells have also been implicated in antilisterial resistance. However, their specific role has remained unclear. Here we show that efficient resistance to infection by this bacterium depends on the functions of both alpha beta and gamma delta T cells in both primary and secondary responses. We also present evidence that these functions are complementary. In the livers of alpha beta T cell-depleted mice, bacteria grow to large numbers within hepatocytes but are infrequently found extracellularly. Granulomatous lesions are more frequent and somewhat larger than in normal controls, but remain focal. Neutrophils are absent from liver lesions in these mice. In contrast, the livers of gamma delta T cell-depleted mice contain many extracellular bacteria, but do not show hepatocytes containing large numbers of Listeria. Liver lesions in gamma delta T cell-depleted mice are far more extensive than in normal controls or in alpha beta T cell-depleted mice, and contain large numbers of neutrophils. Particularly in secondary listeriosis, gamma delta T cell-depleted mice show vast coalescent areas of necrotic liver parenchyma within 48 h after infection. Because the bacterial numbers in gamma delta T cell-depleted mice remain lower than in alpha beta T cell-depleted mice, increased mortality in the former may be in part caused by liver failure. We conclude that gamma delta T cells are required to control inflammatory reactivity and to prevent excessive liver damage during the immune response to Listeria monocytogenes.

Genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB x NZW)F1 mice

Abstract: Lupus-like autoimmunity in (NZB x NZW)F1 mice is frequently marked by the development of a severe and fatal renal disease. Genes from both NZB and NZW parents are required for the full expression of disease. We applied a mapping technique based on polymorphism in simple sequence repeats to the analysis of (NZB x NZW)F1 x NZW backcross mice to determine the NZB genetic contribution to disease. The results show that a single NZB locus or tightly linked group of loci on the distal part of chromosome 4 provides the strongest association with renal disease and death. This locus, designated here as nba-1 (New Zealand Black autoimmunity), lies distal to the locus elp-1, 60-70 centimorgans from the centromere. It is of interest that a gene encoding a receptor for tumor necrosis factor maps to the vicinity of this disease-associated gene.

FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

Abstract: Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants.

Inhibition of IgE production and normalization of airways responsiveness by sensitized CD8 T cells in a mouse model of allergen-induced sensitization

Abstract: The functional role of CD8 T cells in in vivo IgE production, immediate cutaneous reactivity, and altered airways responsiveness (AR) was examined in a murine model of allergen-induced sensitization. Exposure of BALB/c mice to nebulized OVA triggered an IgE anti-OVA response in the serum, immediate-type skin test responses to OVA, and the development of increased AR (as measured by nonspecific reactivity to electrical field stimulation). In spleens of sensitized mice, analysis of the distribution of CD4/CD8 T cell subpopulations revealed an increase in total numbers of CD8 T cells. Transfer of purified spleen CD8 T cells from OVA-sensitized mice (CD8OVA) to sensitized recipients reduced serum IgE anti-OVA production by roughly 50%. Furthermore, studies of in vitro Ig production indicated that mononuclear cells from recipients of CD8 cells (CD8OVA > CD8PBS) produced less IgE and IgG1 antibodies, whereas in vitro IgG2a production was enhanced. The suppression of IgE production in recipients of CD8OVA T cells was associated with the failure to respond to intradermal challenge with OVA. The increase in AR found in sensitized mice was prevented after transfer of CD8OVA cells. When CD8 T cells from nonimmunized animals (CD8PBS) were used for the transfer into sensitized recipients, serum anti-OVA IgE was decreased by only 20%, whereas skin test reactivity and AR were not significantly affected. The ex vivo analysis of the pattern of cytokine-producing lymphocytes by immunofluorescence microscopy indicated that the sensitization procedure increased the fraction of IFN-gamma- and IL-4-positive cells in the spleen. Further in vitro analysis demonstrated that a high percentage of CD8 T cells were positive for IFN-gamma, whereas IL-4 was produced mainly by CD4 T cells. These data suggest that CD8 T cells may play an important role in the negative regulation of IgE production and AR and that IFN-gamma may be a relevant mediator of the functions of CD8 T cells in this model.

Mapping of the C5a receptor signal transduction network in human neutrophils

Abstract: Human neutrophils respond to chemoattractants, resulting in their accumulation at an inflammatory site. Chemoattractants such as the C5a peptide, derived from the C5 complement factor, bind to inhibitory guanine nucleotide binding protein (Gi)-coupled seven membrane-spanning receptors expressed in neutrophils. C5a receptor activation results in the Gi-dependent activation of the mitogen-activated protein (MAP) kinase pathway in human neutrophils. C5a receptor ligation activates both B-Raf and Raf-1, with B-Raf activation overlapping but temporally distinct from that of Raf-1. B-Raf and Raf-1 both efficiently phosphorylate MAP kinase kinase (MEK-1). C5a also stimulates guanine nucleotide exchange and activation of Ras. Ras and Raf activation in response to C5a involves protein kinase C-dependent and -independent pathways. Activation of both Raf-1 and B-Raf was inhibited by protein kinase A stimulation, consistent with the inhibitory effects of increased cAMP levels on neutrophil function. The findings define a functional signal transduction pathway linking the neutrophil C5a chemoattractant receptor to the regulation of Ras, B-Raf, Raf-1, and MAP kinase.

Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes.

Abstract: Stimulation of T cells with antibodies directed towards the T cell receptor complex results in the activation of mitogen-associated protein kinase (MAPK). Two pathways have been described in other cell types that can lead to MAPK activation. One of these pathways involves the activation of Ras, leading to the activation of Raf-1, and the subsequent activation of MEK (MAPK or ERK kinase). The contribution of this pathway in T cells for anti-CD3 or phorbol myristate acetate (PMA)-mediated MAPK activation was examined. We detected the kinase activities of Raf-1 and MEK towards their substrates (MEK for Raf-1 and MAPK for MEK) in this pathway leading to the activation of MAPK. Stimulation of the T cells with either anti-CD3 antibody or PMA resulted in a rapid activation of both Ras and Raf-1. MEK activity towards kinase-active or -inactive recombinant MAPK also increased upon stimulation. In addition, both MAPK and p90rsk were activated in these cells. We suggest that activation of MAPK and the subsequent activation of ribosomal S6 kinase (p90rsk) occurs by the Ras/Raf-1/MEK cascade in T lymphocytes stimulated by ligation of the T cell receptor complex.

Recognition of trophoblasts by gamma delta T cells.

Abstract: The juxtaposition of maternal and fetal tissues in the hemochorial placenta has led to speculation that maternal recognition of fetal Ags present on trophoblasts might play an important role in reproductive biology We report here for the first time such recognition of trophoblasts by T lymphocyte hybridomas representative of certain cells present in the maternal decidua Trophoblast recognition is TCR dependent and is mediated by members of the V gamma 1+ subset of gamma delta T lymphocytes, a population that we previously have shown to be associated with heat shock protein-60 reactivity Recognition occurred in experiments in which trophoblast clones or freshly prepared trophoblasts were used and requires cell-cell interaction Although the maternal-fetal immune relationship typically has been cast in terms of an allograft, the T cell recognition of trophoblasts as described herein is not MHC-restricted, inasmuch as freshly prepared trophoblasts from beta 2-microglobulin-deficient mice were found to be stimulatory Furthermore, the trophoblast ligand that mediates this recognition is probably a conserved mammalian molecule, because a human trophoblast cell line is also stimulatory Our findings suggest a novel form of T cell recognition, and may provide an enhanced understanding of the maternal-fetal immune relationship

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

Abstract: MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

Role of cationic proteins in the airway : hyperresponsiveness due to airway inflammation

Abstract: Major basic protein (MBP) is a highly cationic protein found in the granules of eosinophils. It has been postulated that MBP may participate in the pathogenesis of airway hyperresponsiveness exhibited by asthmatic patients. Accordingly, we have employed a rat system to investigate the effect of human MBP instillation on airway responsiveness and the possible role of cationic charge in the determination of this effect. Major basic protein caused a significant increase in airway responsiveness to inhaled methacholine. Two polycations, poly-L-arginine and poly-L-lysine, also increased airway responsiveness to inhaled methacholine. Moreover, two other very different cationic proteins, platelet factor 4 (PF4) and cathepsin G were also capable of inducing airway hyperresponsiveness. These effects were dependent on their positive charge, since the charge — and, hence the effect — of these proteins was neutralized with low molecular weight heparin. In addition, other polyanions, such as low molecular weight hepar...

Activation of human monocytes via a non-neurokinin substance P receptor that is coupled to Gi protein, calcium, phospholipase D, MAP kinase, and IL-6 production.

Abstract: Substance P (SP) is a tachykinin involved in the regulation of inflammatory processes. Tachykinins bind to three subtypes of neurokinin (NK) receptors. However, recently we demonstrated that monocytes express a SP binding site that is not one of the known NK receptors. Activation of this SP receptor leads to the stimulation of MAP kinase in monocytes. In the present paper we show that this novel SP binding site is coupled to a GTP binding protein of the Gi alpha 1/2 subclass. Triggering of the SP receptor leads to a rapid rise in cytosolic calcium. In a more sustained way, SP stimulates phospholipase D (PLD) activity in human monocytes. The effects of SP on calcium, PLD, and MAP kinase activity can be blocked by pretreatment of the cells with pertussis toxin, which is in agreement with receptor coupling to Gi. At a functional level, stimulation of the non-NK SP receptor on monocytes results in the induction of IL-6 production. We show here that the order of potency for activation of monocytes by various ligands is directly related to the Ki for displacement of labeled SP by these ligands. Therefore, our data strongly suggest that the effects of SP are mediated via the novel SP receptor we recently described.

gp120 ligation of CD4 induces p56lck activation and TCR desensitization independent of TCR tyrosine phosphorylation.

Abstract: HIV gp120 binding to CD4 suppresses TCR function The molecular mechanism of this anergizing effect is incompletely understood Studies reported here reveal that CD4 ligation initiates p56lck activation and renders human peripheral T cells and HPB-ALL cells hyporesponsive to Ag receptor stimulation, as indicated by the failure of TCR binding ligands to induce either protein tyrosine phosphorylation or elevation of intracellular-free calcium concentration To approach the possibility that p56lck-mediated tyrosine phosphorylation of specific sites within TCR zeta-chain might be involved in the gp120-induced TCR signaling defect, we tested the kinase's ability to phosphorylate various zeta peptides Kinetics analyses indicate that peptides derived from the in vitro autophosphorylation site of p56lck Y394 and two sites within zeta, Y84, and Y152, are equally effective substrates for p56lck, whereas p56fyn prefers a substrate peptide derived from a different site within zeta, Y142 Although these data are consistent with the possibility that gp120-mediated signal disruption of TCR could be due to p56lck phosphorylation of Y84 and Y152 residues within zeta, further experiments revealed that gp120 does not induce detectable zeta tyrosine phosphorylation under conditions in which it disrupts TCR signaling These data indicate that gp120 can induce uncoupling of TCR from the earliest events in signal transduction and that this effect can be mediated by a mechanism other than tyrosine phosphorylation of TCR zeta-chain

TCR expression of activated T cell clones in the lungs of patients with pulmonary sarcoidosis.

Abstract: Sarcoidosis is a systemic granulomatous disease of unknown etiology in which CD4+ T cells seem to be critically involved. In the lungs of patients with pulmonary disease, CD4+ T cells accumulate in large numbers, and a subset of these cells is activated. By using both quantitative PCR and anti-V beta mAbs, we analyzed the TCR repertoire of total and activated bronchoalveolar lavage T cells, the latter subset being defined by the ability to proliferate in short-term culture supplemented with IL-2. Overall, there was little difference when TCR V beta expression of freshly isolated lung and peripheral blood cells was compared in individual patients. Some individuals did demonstrate a modest increase in a few V beta-expressing subsets. However, after 1 to 2 wk of in vitro growth in IL-2-supplemented media, bronchoalveolar lavage cells from most patients, but not from any healthy individuals, demonstrated a selective expansion of particular V beta-expressing subsets. Interestingly, different V beta-bearing subsets were expanded in different patients. Junctional region sequencing indicated that the proliferating T cells in culture were strikingly oligoclonal and were derived from T cell clones already selectively expanded in vivo. These results provide evidence for a disease process that involves recognition of local Ag(s) by specific subsets of CD4+ T cells. Analysis of the Ag specificity of these IL-2-expanded populations is likely to provide insight into the pathogenesis of this disease.

How does the G protein, Gi2, transduce mitogenic signals?

Abstract: Serpentine receptors coupled to the heterotrimeric G protein, Gi2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the Gi2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the Gi2-coupled thrombin and acetylcholine muscarinic M2 receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. Gi2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of αi2 inhibits both the Raf-dependent and-independent pathways activated by Gi2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by Gi2-coupled receptors as well as other receptor systems, including the tyrosine kinases.

Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C.

Abstract: Mitogen-activated protein kinase cascades are conserved in fungal, plant, and metazoan species. We expressed murine MAP kinase kinase kinase (MEKK) in the yeast Saccharomyces cerevisiae to determine whether this kinase functions as a general or specific activator of genetically and physiologically distinct MAP-kinase-dependent signaling pathways and to investigate how MEKK is regulated. Expression of MEKK failed to correct the mating deficiency of a ste11 delta mutant that lacks an MEKK homolog required for mating. MEKK expression also failed to induce expression of a reporter gene controlled by the HOG1 gene product (Hog1p), a yeast MAP kinase homolog involved in response to osmotic stress. Expression of MEKK did correct the cell lysis defect of a bck1 delta mutant that lacks an MEKK homolog required for cell-wall assembly. MEKK required the downstream MAP kinase homolog in the BCK1-dependent pathway, demonstrating that it functionally replaces the BCK1 gene product (Bck1p) rather than bypassing the pathway. MEKK therefore selectively activates one of three distinct MAP-kinase-dependent pathways. Possible explanations for this selectivity are discussed. Expression of the MEKK catalytic domain, but not the full-length molecule, corrected the cell-lysis defect of a pkc1 delta mutant that lacks a protein kinase C homolog that functions upstream of Bck1p. MEKK therefore functions downstream of the PKC1 gene product (Pkc1p). The N-terminal noncatalytic domain of MEKK, which contains several consensus protein kinase C phosphorylation sites, may, therefore, function as a negative regulatory domain. Protein kinase C phosphorylation may provide one mechanism for activating MEKK.

Oral desensitization to rifampin and ethambutol in mycobacterial disease.

Abstract: The incidence of disease caused by Mycobacterium tuberculosis (including drug-resistant strains) and M. avium complex (MAC) is increasing. Hypersensitivity reactions to antimycobacterial agents are relatively uncommon, but when they occur they may result in cessation of therapeutic medications. We report our experience with rapid oral desensitization to ethambutol and rifampin in a group of 10 patients with mycobacterial disease who had experienced cutaneous hypersensitivity reactions to these drugs. An adaptation of the rapid oral desensitization protocol for penicillin was used, with the dosing intervals increased to account for the different kinetics of these drugs. Adverse reactions were few and easily treated without necessitating cessation of therapy. We conclude that oral desensitization to rifampin and ethambutol by our protocol is safe and effective, allowing these patients to proceed with an optimal antimycobacterial regimen.

Structural requirements for peptides that stimulate a subset of gamma delta T cells.

Abstract: Hybridomas representing the V gamma 1-positive subset of murine gamma delta T cells secrete lymphokines in response to synthetic peptides representing a short segment of the mycobacterial 60-kDa heat shock protein (HSP-60). Here we show the TCR dependency of this response by transfection of productively rearranged TCR genes derived from an HSP-60 reactive gamma delta T cell hybridoma. We also have defined structural requirements for the stimulatory peptide. The smallest HSP-60 peptide capable of stimulating these hybridomas is seven amino acids long, representing positions 181-187, and having the sequence FGLQLEL. Amino acid-substituted derivatives of this peptide, and another containing the same core, p180-190, revealed amino acids essential for stimulatory activity. Phenylalanine in position 181 and leucine in position 183 seem to be required for stimulation of all HSP-60 reactive cells, whereas others are only required by some. Clonal differences in the responses to these peptides provide indirect evidence for cognate TCR-peptide interactions. The smallest stimulatory peptide, p181-187, represents an area not well conserved among HSP-60 molecules of other species, and stimulates a mycobacteria-specific response unlike the earlier observed cross-reactive responses of the same hybridomas with longer HSP-60 peptides derived from mycobacteria and other species (our manuscript in preparation). We propose that the TCR-dependent multiclonal gamma delta T cell response to HSP-60 peptides and derivatives, which in some ways resembles superantigen responses and in other ways resembles responses to conventional Ag, may be a separate, third type of Ag response by T cells.

Evidence for polyclonal T cell activation in murine models of systemic lupus erythematosus.

Abstract: CD4+ T cells have been shown to be important in the development of disease in murine models of SLE. We compared the TCR V beta repertoires of young (healthy) and older (diseased) New Zealand hybrid mice as well as non-autoimmune strains to characterize changes in TCR usage associated with the development of disease. Despite large increases in the total number of splenic CD4+ T cells with age in diseased mice, we noted little skewing of the V beta repertoire. For example, diseased NZB.H-2bm12 mice failed to exhibit a significant change in the percentage of any V beta subset despite a fivefold increase in the number of CD4+ T cells. Strains without lupus-like disease, including NZB.H-2b mice, demonstrated no increase in CD4+ T cell numbers with age. Similar to NZB.H-2bm12 mice, (NZB x SWR)F, and (NZB x NZW)F1 mice showed disease-related increases in CD4+ T cell numbers, but no changes in V beta repertoire that could be linked to disease development. Differences in V beta usage between young autoimmune and non-autoimmune strains of mice matched for either MHC or background genes were consistent with genetic influences unrelated to disease. Overall, the heterogeneous repertoire of proliferating T cells provides evidence for polyclonal T cell expansion in murine models of lupus and suggests that activation either involves a multitude of conventional self-antigens or may be independent of the TCR. However, the requirement for specific class II MHC molecules suggests that this polyclonal T cell expansion is dependent on a much smaller and specific autoreactive response.

Monocytes express a non-neurokinin substance P receptor that is functionally coupled to MAP kinase.

Abstract: The data presented in this paper demonstrate a new substance P (SP) binding site that is expressed on human monocytes. The apparent dissociation constant (Kd) for binding of 125I-labeled Bolton Hunter-SP (125I-BH-SP) to the receptor on monocyte membranes is 2.24 +/- 0.9 x 10(-7) M and the maximum binding capacity (Bmax) is 4.7 +/- 0.5 pmol/mg membrane protein. It could be excluded that this receptor is one of the known neurokinin (NK) type of receptors on the basis of binding characteristics for NK1, NK2, and NK3 agonists. Moreover, we demonstrate that the binding site is neither the bombesin receptor nor the serpin enzyme complex receptor nor the FMLP receptor. The order of potency for inhibition of 125I-BH-SP binding to the receptor on monocyte membranes is NK1 antagonist [D-Pro2,D-Trp7,9]SP > SP > NK3 agonist [MePhe7]SP > bombesin. Cross-linking studies with disuccinimidylsuberate, followed by SDS-PAGE analysis, revealed that 125I-BH-SP is specifically bound to a membrane protein with an apparent molecular mass of 47 kDa. At a functional level, SP induces the activation of MAP kinase in human monocytes. The ED50 for activation of MAP kinase positively correlated (r = 0.999, p < 0.0005) with the apparent affinity of the ligands applied in the 125I-BH-SP displacement studies. From these results, we conclude that this SP binding site on monocytes is a non-NK receptor protein that is functionally linked to the activation of MAP kinase.

Neutrophils increase volume during migration in vivo and in vitro.

Abstract: Neutrophils increase volume (approximately 15%) when stimulated in suspension, but whether a similar alteration occurs in vivo during migration is unknown. We measured neutrophil volume using serial 0.5-micron sections and three-dimensional reconstruction of rabbit neutrophils migrating into inflammatory lesions in lung and abdominal wall in vivo and of human neutrophils migrating across collagen gels in vitro. An inflammatory response was induced by local instillation of C5a in vivo or generating a gradient of FMLP in vitro. In the lung, neutrophils reconstructed within the vascular space, either in arterioles (158 microns3), capillaries (128 microns3), or venules (135 microns3), were of similar volume, while those in the airspace were markedly larger (266 microns3). Neutrophils that migrated into the abdominal wall (150 microns3) were also significantly larger than those in the abdominal wall vasculature (100 microns3). Human neutrophils induced to migrate into collagen gels by FMLP were significantly larger (290 microns3) than those that did not migrate (204 microns3). We conclude from these studies that migration of rabbit neutrophils in vivo or human neutrophils in vitro is associated with a substantial increase in volume. We speculate that these findings hold promise for elucidation of the mechanisms of neutrophil migration.

Maturation of nonadrenergic noncholinergic inhibitory system in normal and allergen-sensitized rabbits.

Abstract: We investigated the functional existence of the nonadrenergic noncholinergic inhibitory (NANCi) system in developing rabbit airways in vitro. Furthermore, we evaluated the effect of parenteral exposure to a specific allergen (ragweed) on the maturation of this neural pathway. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from normal and ragweed-sensitized New Zealand White rabbits at 1, 2, 4, and 12 wk of age. The TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit solution, atropine (1 x 10(-5) M), and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation was applied at stimulation frequencies ranging from 5 to 30 Hz to determine the frequency that produced maximal relaxation. The NANCi response to EFS was measured and expressed as the mean (+/- SE) percent relaxation at 20 Hz, because this stimulation frequency gave the maximal NANCi response at each age studied. TSM segments obtained from control rabbits at 1 wk of age did not demonstrate a NANCi response at the frequencies of stimulation used. By contrast, a reproducible NANC relaxation was demonstrated in TSM from 2-, 4-, and 12-wk-old rabbits. The magnitude of this response was 27 +/- 4.7 (n = 10), 29 +/- 4.8 (n = 9), and 37 +/- 4% (n = 18), respectively. The same experiments performed on TSM segments obtained from ragweed-sensitized animals gave significantly decreased values of NANCi response. In 2-, 4-, and 12-wk-old rabbits, the NANCi responses were 11.5 +/- 3.4 (n = 9), 11 +/- 2 (n = 13), and 16 +/- 4.2% (n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of MAP2-kinase in B lymphocytes by calcium ionophores.

Abstract: The role of increases in intracellular calcium levels on tyrosine phosphorylation in human B lymphocytes was studied. Stimulation of normal, resting B lymphocytes or B lymphoblastoid cells with the calcium ionophores ionomycin or A23187 induced the tyrosine phosphorylation and the enzymatic activation of microtubule-associated protein-2 kinase (MAP2-K). Treatment of these cells with PMA induced tyrosine phosphorylation of a protein with the identical mobility, as well as the enzymatic activation of MAP2-K. Stimulation of these cells with ionomycin also resulted in increased ribosomal S6 kinase activity. Activation of MAP2-K in B lymphocytes by calcium ionophore was rapid (detectable within 1 min), transient (returning to background levels by 45 min), and dependent on extracellular calcium. These results demonstrate that transmembrane calcium flux induced by calcium ionophore results in the tyrosine phosphorylation and enzymatic activation of MAP2-K in human B cells.

Autocrine stimulation of B lymphocytes by a platelet-activating factor receptor agonist, 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine.

Abstract: Based on previous data which demonstrated the ability of platelet-activating factor (PAF) antagonists to inhibit constitutive immunoglobulin synthesis in B-lymphoblastoid cell lines, we determined the capacity of these cells to synthesize PAF or 1-palmitoyl-2-acetyl-sn-glycero-phosphocholine (PAGPC), an acyl-PAF identified in various cell types. In two B-lymphoblastoid cell lines (LA350 and HSCE-), significant amounts of production of PAGPC were detected, whereas the amount of PAF was below the level of detection in our system. The biologic effects of PAGPC were examined in these cells, both of which have well-characterized PAF receptors. PAGPC induced a concentration-dependent increase in intracellular Ca2+ concentrations and activation of MAP-2 kinase (as detected by immunoblotting and measurements of kinase activity) in these cells. The kinetics and magnitude of these responses were similar to those induced by PAF, and they were inhibited by Web 2086, a PAFR antagonist. Phosphatidylcholine, which differs from PAGPC in that it contains a long fatty acid residue at position 2, did not induce any of these responses. A mutual cross-desensitization of the B lymphoblasts between PAGPC and PAF was observed for Ca2+ mobilization. To induce maximal cell stimulation, approximately 600-fold higher concentrations of PAGPC than of PAF were needed. Because the two B-lymphoblastoid cell lines synthesized significant amounts of PAGPC, this phospholipid may participate in an autocrine stimulation pathway in B cells. Furthermore, the data indicate that PAGPC is an agonist for the PAFR in B lymphoblasts and that the ether linkage in the PAF molecule is not an absolute requirement for activity, although it increases the potency of the ligand.

Isolation of a gene encoding a novel receptor tyrosine kinase from differentiated embryonic stem cells.

Abstract: A mouse gene encoding a receptor tyrosine kinase, designated Embryonic receptor kinase (EmRK2), was isolated from embryoid bodies (EBs) generated by differentiating embryonic stem (ES) cells in culture for 6 days. Sequence analysis of EmRK2 cDNA clones predicts a receptor with a 755 amino acid extracellular region with seven immunoglobulin-like domains, a transmembrane region, and a 552 amino acid cytoplasmic region containing the kinase domain. The kinase domain is interrupted by a stretch of hydrophilic amino acids, the kinase insert. EmRK2 is expressed in embryoid bodies, in whole embryos at day 10 and 12 of gestation, and in the embryonic yolk sac and the fetal liver. On the basis of sequence homology, EmRK2 is likely to be the mouse homologue of human flt, a receptor for vascular endothelial growth factor, and as such, could encode an endothelial cell specific receptor tyrosine kinase.

Allelic differences in TCR gamma-chains alter gamma delta T cell antigen reactivity.

Abstract: Mouse gamma delta T cell hybridomas from various strains that express a TCR-V gamma 1/V delta 6 respond weakly to an autologous Ag and more strongly to a short segment of the mycobacterial heat shock protein-60 (HSP-60). However, V gamma 1/V delta 6 hybridomas derived from AKR mice show greatly reduced or absent responses to these stimuli. To determine whether the lack of response in these AKR hybridomas is caused by polymorphisms found in the expressed AKR gamma and TCR-delta genes or, instead, stems from other genes in AKR, we crossed an AKR mouse with a responder mouse, C57BL/10 (B10), and prepared hybridomas from F1 progeny. Expression of an AKR V gamma 1-J gamma 4-C gamma 4 gene correlated with nonresponsiveness, whereas expression of a B10 V gamma 1-J gamma 4-C gamma 4 gene in most hybridomas ensured responses to both self Ag and the HSP-60 peptide. An allelic difference in the expressed V gamma 6 gene was irrelevant to these responses. Moreover, transfection of a functional B10 V gamma 1-J gamma 4-C gamma 4 gene into an F1 hybridoma variant that had lost the AKR version of this gene restored responses. The allelic gamma gene products differ at nine amino acids in the V region, and three amino acids in the C region. In addition, the AKR C gamma 4 region contains a 16-amino acid insertion. We propose that amino acid differences among those encoded by the AKR V gamma 1-J gamma 4-C gamma 4 gene are responsible for the lack of response, and reduce the ability of the TCR-gamma delta to bind the relevant Ag.