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For activation T cells engage antigen-presenting cells (APCs) in lymphatic tissues. The contact duration and kinetics (static versus dynamic) vary considerably in different model systems; however, it is unclear whether T cells, APCs, or the environment are responsible for the observed discrepancies. Using 3-D collagen matrices as structural scaffold, we directly compared the kinetics of T-cell engagement and activation by functionally major APC types, ie, dendritic cells (DCs) and resting or activated B cells. Resting B cells engaged T cells in long-lived (several hours), adhesive, and leukocyte function-associated antigen-1 (LFA-1)-dependent conjugates in 3-D collagen as well as in intact lymph nodes in vivo. DCs and preactivated B cells, however, supported predominantly dynamic, short-lived (minutes), and sequential contacts to T cells that were dependent on high cytoskeletal activity of the APCs but could not be inhibited by anti-LFA-1 treatment. Naive T cells were most strongly activated by DCs and activated B cells, whereas resting B cells were 100-fold less efficient to induce T-cell proliferation. Thus, in the same 3-D environment, naive T cells respond with a spectrum of different interaction modes dependent on the type and activation state of the APCs. Thereby, more dynamic interaction kinetics is positively correlated with higher T-cell priming efficiency.

Cytohesins are a family of highly homologous guanine nucleotide exchange factors (GEFs) that act on ADP-ribosylation factors (ARFs). The small ARF-GEFs are involved in integrin signaling, actin cytoskeleton remodeling, and vesicle transport. Here, we selected and applied a specific inhibitor for ARF nucleotide-binding site opener (ARNO)/cytohesin-2, an RNA aptamer that clearly discriminates between cytohesin-1 and cytohesin-2. This reagent bound to an N-terminal segment of cytohesin-2 and did not inhibit ARF-GEF function in vitro. When transfected into HeLa cells, it persisted for at least 6 h without requiring stabilization. Its effect in vivo was to down-regulate gene expression mediated through the serum-response element and knockdown mitogen-activated protein kinase activation, indicating that cytohesin-2 acts by means of mitogen-activated protein kinase signaling. We conclude that the N-terminal coiled-coil and parts of the Sec7 domain of cytohesin-2 are required for serum-mediated transcriptional activation in nonimmune cells, whereas cytohesin-1 is not. Our results indicate that intramer technology can be used not only for assigning novel biological functions to proteins or protein domains but also to prove nonredundancy of highly homologous proteins.

The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.

An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions.

Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.

Endothelial migration on extracellular matrix is regulated by integrins and proteolysis. Previous studies showed that beta(3)-integrins regulate expression of the urokinase-type plasminogen activator receptor (uPAR) through outside-in signalling involving the cytoplasmic domain. Here we show that overexpression of the integrin-binding protein beta(3)-endonexin decreased uPAR promoter (-398 base-pair fragment) activity that is constitutively active in endothelial cells. Mutation of the NF-kappaB promoter binding site (-45 bp) impaired the ability of beta(3)-endonexin to downregulate uPAR promoter activity. Immunoprecipitation studies showed that beta(3)-endonexin interacts directly with the p50/p65 transactivation complex and thereby inhibits binding of kappaB oligonucleotides to the p50/p65 complex. Moreover, binding of beta(3)-endonexin to p50 was inhibited in the presence of kappaB but not mutated kappaB oligonucleotides, suggesting a sterical competition between beta(3)-endonexin and kappaB DNA for the p50/p65 complex. We therefore propose that beta(3)-endonexin acts as regulator of uPAR expression in beta(3)-integrin-mediated endothelial cell migration through direct interaction with p50/p65. Since NF-kappaB regulates the expression of matrix degrading enzymes, the present results define a role of beta(3)-endonexin in regulating beta(3)-integrin-mediated adhesion and pericellular proteolysis.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.

Cytohesin-1 is a regulatory interaction partner of the beta2 integrin alphaLbeta2 (LFA-1) and a guanine exchange factor (GEF) for ADP ribosylation factor (ARF)-GTPases. However, a functional role of cytohesin-1 in leukocyte adhesion to activated endothelium and subsequent transmigration in response to chemokines has not been defined. Overexpression of cytohesin-1 increased LFA-1-dependent arrest of leukocytic cells triggered by chemokines on cytokine-activated endothelium in flow while reducing the fraction of rolling cells. Conversely, a dominant-negative PH domain construct of cytohesin-1 but not a mutant deficient in GEF activity impaired arrest, indicating an involvement of the PH domain while GEF function is not required. Expression of these constructs and a beta2 mutant interrupting the interaction with cytohesin-1 indicated that shape change in flow and transendothelial chemotaxis involve both LFA-1 avidity regulation and GEF activity of cytohesin-1. As a potential downstream target, ARF6 but not ARF1 was identified to participate in chemotaxis. Our data suggest that cytohesin-1 and ARF6 are involved in the dynamic regulation of complex signaling pathways and cytoskeletal remodeling processes governing LFA-1 functions in leukocyte recruitment. Differential effects of cytohesin-1 and ARF6 mutants in our systems reveal that cytohesin-1 with its GEF activity controls both conversion of rolling into firm arrest and transmigration triggered by chemokines, whereas a cyclical activity of ARF6 plays a more important role in diapedesis.

Cell adhesion mediated by integrin receptors is controlled by intracellular signal transduction cascades. Cytohesin-1 is an integrin-binding protein and guanine nucleotide exchange factor that activates binding of the leukocyte integrin leukocyte function antigen-1 to its ligand, intercellular adhesion molecule 1. Cytohesin-1 bears a carboxyl-terminal pleckstrin homology domain that aids in reversible membrane recruitment and functional regulation of the protein. Although phosphoinositide-dependent membrane attachment of cytohesin-1 is mediated primarily by the pleckstrin homology domain, this function is further strengthened by a short carboxyl-terminal polybasic amino acid sequence. We show here that a serine/threonine motif within the short polybasic stretch of cytohesin-1 is phosphorylated by purified protein kinase C delta in vitro. Furthermore, the respective residues are also found to be phosphorylated after phorbol ester stimulation in vivo. Biochemical and functional analyses show that phosphorylated cytohesin-1 is able to tightly associate with the actin cytoskeleton, and we further demonstrate that phosphorylation of the protein is required for maximal leukocyte function antigen-1-mediated adhesion of Jurkat cells to intercellular adhesion molecule 1. These data suggest that both phosphatidylinositol 3-kinase and protein kinase C-dependent intracellular pathways that stimulate beta(2)-integrin-mediated adhesion of T lymphocytes converge on cytohesin-1 as functional integrator.

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

The induction of a transformed cellular phenotype by viruses requires the modulation of signaling pathways through viral proteins. We show here that the phenotypic changes induced by the kaposin A protein of human herpesvirus 8 are mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion. Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. Furthermore, liposome-embedded kaposin A specifically stimulates cytohesin-1 dependent GTP binding of myristoylated ARF1 in vitro. These results suggest a previously unknown involvement of ARF GTPases in the control of cellular functions by herpesviruses.

CD4 recruitment to T cell receptor (TCR)-peptide-major histocompatibility class II complexes is required for stabilization of low affinity antigen recognition by T lymphocytes. The cytoplasmic portion of CD4 is thought to amplify TCR-initiated signal transduction via its association with the protein tyrosine kinase p56(lck). Here we describe a novel functional determinant in the cytosolic tail of CD4 that inhibits TCR-induced T cell activation. Deletion of two conserved hydrophobic amino acids from the CD4 carboxyl terminus resulted in a pronounced enhancement of CD4-mediated T cell costimulation. This effect was observed in the presence or absence of p56(lck), implying involvement of alternative cytosolic ligands of CD4. A two-hybrid screen with the intracellular portion of CD4 identified a previously unknown 33-kDa protein, ACP33 (acidic cluster protein 33), as a novel intracellular binding partner of CD4. Since interaction with ACP33 is abolished by deletion of the hydrophobic CD4 C-terminal amino acids mediating repression of T cell activation, we propose that ACP33 modulates the stimulatory activity of CD4. Furthermore, we demonstrate that interaction with CD4 is mediated by the noncatalytic alpha/beta hydrolase fold domain of ACP33. This suggests a previously unrecognized function for alpha/beta hydrolase fold domains as a peptide binding module mediating protein-protein interactions.

ADP-ribosylation factors (ARFs) are small Ras-like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin-1, a 47-kDa cytoplasmic protein acts as an inside-out signaling molecule and regulates binding of the beta2 integrin leukocyte function antigen 1 (LFA-1) to its ligand intercellular adhesion molecule 1 (ICAM-1). In this study, we address the regulation of the GEF activity of cytohesin-1 by phosphoinositides, using mammalian expression of functional ARF-Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A-Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1-Ig and ARF6-Ig chimeras are activated in vitro by cytohesin-1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.

Intracellular signaling pathways, which regulate the interactions of integrins with their ligands, affect a wide variety of biological functions. Here we provide evidence of how cytohesin-1, an integrin-binding protein and guanine-nucleotide exchange factor (GEF) for ARF GTPases, regulates cell adhesion. Mutational analyses of the beta-2 cytoplasmic domain revealed that the adhesive function of LFA-1 depends on its interaction with cytohesin-1, unless the integrin is activated by exogenous divalent cations. Secondly, cytohesin-1 induces expression of an extracellular activation epitope of LFA-1, and the exchange factor function is not essential for this activity. In contrast, LFA-1-mediated cell adhesion and spreading on intercellular cell adhesion molecule 1 is strongly inhibited by a cytohesin-1 mutant, which fails to catalyze ARF GDP-GTP exchange in vitro. Thus, cytohesin-1 is involved in the activation of LFA-1, most probably through direct interaction with the integrin, and induces cell spreading by its ARF-GEF activity. We therefore propose that both direct regulation of the integrin and concomitant changes in the membrane topology of adherent T cells are modulated by dissectable functions of cytohesin-1.

It has been proposed that the maintenance of T cell anergy depends on the induction of negative regulatory factors. Differential display of reverse transcribed RNA was used to identify novel genes that might mediate this function in anergic Th1 clones. We report that anergic Th1 clones do indeed express a genetic program different from that of responsive T cells. Moreover, one gene, the general receptor of phosphoinositides 1 (GRP1), was selectively induced in anergic T cells. The GRP1, located in the plasma membrane, regulated integrin-mediated adhesion and was invariably associated with unresponsiveness in multiple models of anergy. T cells expressing retrovirally transduced GRP1 exhibited normal proliferation and cytokine production. However, GRP1-transduced T cells were not stable and rapidly lost GRP1 expression. Thus, although GRP1 may not directly mediate T cell anergy, it regulates cell expansion and survival, perhaps through its integrin-associated activities.

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the beta2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in approximately 100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2-3 microM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the beta-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation-isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton's tyrosine kinase with the adjacent Bruton's tyrosine kinase motif, a novel zinc-containing fold.

The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor-receptor (TNF-R55) appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By combined use of the yeast interaction trap system and a peptide scanning library, the novel WD-repeat protein FAN has been identified, which specifically binds to a cytoplasmic nine amino acid binding motif of TNF-R55. This region has been previously recognized as a distinct functional domain that is both required and sufficient for the activation of neutral sphingomyelinase (N-SMase). Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, while truncated mutants of FAN produced dominant negative effects. The data suggest that FAN regulates ceramide production by N-SMase, which is a crucial step in TNF signaling.

The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated. We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18). The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain. Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion. Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.

TcR1+ T cells in peripheral blood have been shown to express in an exclusive fashion either the Ti gamma A or the delta TCS1 epitope. Here, we characterize a subset of TcR1+ T cells in fresh thymus co-expressing the Ti gamma A and delta TCS1 epitopes. TcR1dim and TcR1bright clones can be distinguished. Biochemical and molecular studies on both types of clones generated from these thymocytes reveal a unique T cell receptor structure characterized by the use of a V gamma 9/C gamma 2-encoded 55-kDa gamma chain nondisulfide linked to a V delta 1-encoded delta chain.

CD62, also called PADGEM protein, GMP-140, or P-selectin, is a granule membrane protein of endothelial cells and platelets that is mobilized to the plasma membrane following exposure to mediators such as thrombin, histamine, complement components, or peroxides. Data presented to date suggest that one ligand of CD62 includes CD15 (Lewis x determinant) and sialic acid. We show here that sulfatides, heterogeneous 3-sulfated galactosyl ceramides, are an apparently unrelated ligand of CD62. Sulfatides are expressed on the plasma membrane of, and are excreted by, granulocytes, and constitute the principal ligand for CD62 on the plasma membrane of some tumor cells. CD62 binds to sulfatides adsorbed to plastic as avidly as it binds to myeloid or tumor cells. We find that granulocytes excrete sulfatides at a rate predicted to allow them to be rapidly released from CD62 once they have exited the bloodstream.

The role of natural killer (NK) cells in rheumatoid arthritis (RA) remains unclear. A pathogenetic function of rheumatoid factors (RF) also has not been defined. In the present studies, natural killer (NK) cells were examined as a model for FC gamma receptor type III-positive (FC gamma RIII+) cells, with regard to their interaction with RF. NK cell antigen CD16 (FC gamma RIII) and CD56 expression and functional NK and antibody-dependent cell-mediated cytotoxicity (ADCC) activity were compared in peripheral blood lymphocytes and autologous synovial fluid lymphocytes (SFL) of RA patients. Peripheral blood lymphocytes and SFL showed normal CD56 expression. In contrast, both the frequency and the density of CD16 antigen were decreased in SFL. Furthermore, diminished NK cytotoxicity and a significant decrease in ADCC were observed in SF NK cells. In subsequent in vitro studies with normal fresh NK cells, it was demonstrated that IgG-containing RF complexes from RA patients induced a modulation of FC gamma RIII structure from the NK cell surface, a decrease in NK activity, and a complete loss of ADCC. When purified RF was incubated with NK-enriched cell lines from RA patients, increased transcription and subsequent production of interferon-gamma and tumor necrosis factor alpha were observed. These data suggest a direct involvement of RF complexes in the pathogenetic process of chronic inflammation in RA.

Endothelial leukocyte adhesion molecule-1 (ELAM-1) is an endothelial cell adhesion molecule that allows myeloid cells to attach to the walls of blood vessels adjacent to sites of inflammation. ELAM-1 recognizes the sialyl-Lewis X (sialyl-Lex) determinant, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, a granulocyte carbohydrate also found on the surface of some tumor cell lines. Binding of myeloid cells to soluble ELAM-1 is inhibited by a monoclonal antibody recognizing sialyl-Lex or by proteins bearing sialyl-Lex, some of which may participate in humoral regulation of myeloid cell adhesion. Stimulated granulocytes also release an inhibitor of ELAM-1 binding that can be selectively adsorbed by monoclonal antibody to sialyl-Lex.

Fc gamma-receptor-(CD16-)positive natural killer (NK) cells are activated by rheumatoid factor in the synovial fluid of patients with rheumatoid arthritis. This activation induces modulation of the CD16 molecule and release of IFN gamma and TNF alpha by NK cells.

Pulmonary infection with mycobacterium tuberculosis and clonal B-cell expansion is described in a 26-year-old woman with granulomatous disease of lung, liver, and bone marrow as well as a late onset common variable immunodeficiency syndrome (CVID).

A nuclear gene encoding a light-induced transiently expressed protein that is localized in the chloroplast has been isolated from an EMBL3 library of pea DNA. The gene is a member of a multigene family. The sequence of the gene contains the complete reading frame of previously characterized cDNA clones and two introns in the 5' region of the protein coding sequence. Primer extension and S1 nuclease studies have defined the cap site. Two TATA boxes are found 5' to the initiating methionine codon. Only a limited homology is found between the presequence of the gene and transit sequences of other previously sequenced precursors. Isolated nuclei of pea have been labelled and the in vitro synthesized transcripts analysed. The results show that the light-dependent expression of the gene family is regulated at the level of transcription.

cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2-4 h after onset of illumination in five day old, etiolated pea seedlings.The cDNA library was constructed from poly(A)(+)-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with(32)P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of Mr 24 000. After posttranslational importin vitro, the processed product of Mr 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2-4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.

Fc receptor-bound human immunoglobulin G can be detected in vitro and in vivo on CD16+ natural killer cells. Cross-linking of CD16-bound IgG via monoclonal antibody results in increased cytotoxic activity of effector cells.