Recruitment of SLP-76 to the membrane and glycolipid-enriched membrane microdomains replaces the requirement for linker for activation of T cells in T cell receptor signaling.

Two hematopoietic-specific adapters, src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224-244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cgamma1 phosphorylation, extracellular signal-regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224-244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

In vitro and in vivo macrophage function can occur independently of SLP-76.

Expression of SH2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), a hematopoietic cell-specific adapter protein, is required to couple Syk family tyrosine kinase activation to downstream mediators such as phospholipase C (PLC)-gamma following TCR, platelet collagen receptor and mast cell Fc epsilon R stimulation. In addition to T cells, mast cells and platelets, SLP-76 is expressed in monocytes and macrophages. To determine the role of SLP-76 in Fc gamma R-stimulated signaling pathways in macrophages, we examined cultured bone marrow-derived macrophages (BMM) from SLP-76(-/-) and wild-type mice. In this study, we show that Fc gamma R cross-linking rapidly induces tyrosine phosphorylation of SLP-76 in wild-type BMM. Surprisingly, however, BMM from SLP-76(-/-) mice activate ERK2 and phosphorylate PLC-gamma 2 following Fc gamma R ligation. Furthermore, SLP-76(-/-) BMM display normal Fc gamma R-dependent phagocytic function and reactive oxygen intermediate production. SLP-76(-/-) and SLP-76(+/+) BMM secrete comparable levels of IL-12 in response to lipopolysaccharide and IFN-gamma. To examine macrophage function in vivo, SLP-76(-/-) mice were challenged i.v. with Listeria monocytogenes. SLP-76(-/-) mice survive and efficiently contain the acute phase of infection similar to wild-type mice but exhibit a stable chronic infection attributed to the lack of mature T cells. These data show that, although SLP-76 is required to couple Syk family PTK activity to downstream mediators and effector functions in Fc gamma R-induced pathways in some cell types, activation of Fc gamma R-dependent pathways occurs independently of SLP-76 in BM

NK cytokine secretion and cytotoxicity occur independently of the SLP-76 adaptor protein.

The adapter protein SLP-76 is required for T cell development and TCR signal transduction. SLP-76 is also expressed in NK cells, yet splenic populations of NK cells develop normally in SLP-76-deficient mice. We examined the effects of SLP-76 deficiency upon cellular activation through studies of NK function in SLP-76(-/-) mice. This study presents evidence that NK populations in both spleen and liver of SLP-76(-/-) mice remain intact. Natural cytotoxic responses of SLP-76(-/-) splenocytes proceed in a manner comparable to those of wild-type control splenocytes. Similar to controls, SLP-76(-/-) splenocytes exhibit enhanced survival and augmented cytotoxic capacity after in vitro culture with IL-2. IL-2-stimulated SLP-76(-/-) splenocytes also retain normal antibody-dependent cellular cytotoxicity and the ability to secrete IFN-gamma in response to IL-12 stimulation. These results indicate that, unlike events stimulated by TCR engagement, signaling cascades engaged by IL-2 and IL-12 receptors, by Fc gammaRIIIA (which mediates antibody-dependent cellular cytotoxicity), and by natural cytotoxicity-associated receptors on murine NK cells can occur independently of SLP-76.

Integration of T cell receptor-dependent signaling pathways by adapter proteins.

The initiation of biochemical signal transduction following ligation of surface receptors with intrinsic cytoplasmic tyrosine kinase activity is common for many cell types. T lymphocytes also require activation of tyrosine kinases following T cell receptor (TCR) ligation for maximal stimulation. However, the TCR has no intrinsic tyrosine kinase activity. Instead, the TCR must rely on cytoplasmic tyrosine kinases that localize to the TCR complex and initiate TCR-mediated signaling events. Although much has been learned regarding how these cytosolic tyrosine kinases are activated and recruited to the TCR complex, relatively little is understood about how these initial events are translated into transcriptional activation of genes that regulate cytokine production, cell proliferation, and cell death. Recently, it has become clear that the class of intracellular molecules known collectively as adapter proteins, molecules with modular domains capable of recruiting additional proteins but that exhibit no intrinsic enzymatic activity, serve to couple proximal biochemical events initiated by TCR ligation with more distal signaling pathways.

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets.

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.

Fetal hemorrhage and platelet dysfunction in SLP-76-deficient mice.

The adapter protein SLP-76 is expressed in T lymphocytes and hematopoietic cells of the myeloid lineage, and is known to be a substrate of the protein tyrosine kinases that are activated after ligation of the T-cell antigen receptor. Transient overexpression of SLP-76 in a T-cell line potentiates transcriptional activation after T-cell receptor ligation, while loss of SLP-76 expression abrogates several T-cell receptor-dependent signaling pathways. Mutant mice that lack SLP-76 manifest a severe block at an early stage of thymocyte development, implicating SLP-76 in signaling events that promote thymocyte maturation. While it is clear that SLP-76 plays a key role in development and activation of T lymphocytes, relatively little is understood regarding its role in transducing signals initiated after receptor ligation in other hematopoietic cell types. In this report, we describe fetal hemorrhage and perinatal mortality in SLP-76-deficient mice. Although megakaryocyte and platelet development proceeds normally in the absence of SLP-76, collagen-induced platelet aggregation and granule release is markedly impaired. Furthermore, treatment of SLP-76-deficient platelets with collagen fails to elicit tyrosine phosphorylation of phospholipase C-gamma2 (PLC-gamma2), suggesting that SLP-76 functions upstream of PLC-gamma2 activation. These data provide one potential mechanism for the fetal hemorrhage observed in SLP-76-deficient mice and reveal that SLP-76 expression is required for optimal receptor-mediated signal transduction in platelets as well as T lymphocytes.

SLP-76 expression is restricted to hemopoietic cells of monocyte, granulocyte, and T lymphocyte lineage and is regulated during T cell maturation and activation.

The leukocyte-specific adapter protein SLP-76 is known to augment the transcriptional activity of nuclear factor of activated T cells and AP-1 following TCR ligation. A role for SLP-76 in additional receptor-mediated signaling events is less clear. To define the pattern of SLP-76 expression during murine hemopoiesis, we stained cells isolated from various tissues with a combination of surface markers followed by intracellular staining with a fluorochrome-labeled SLP-76-specific Ab. In the bone marrow, SLP-76 expression is largely restricted to cells of granulocyte and monocyte lineage. Heterogeneous SLP-76 expression is first detected in the CD44+ CD25- subset within the CD3- CD4- CD8- thymocyte population. Interestingly, SLP-76 expression increases as thymocyte maturation progresses within the CD4- CD8- compartment but decreases as cells mature to a CD4+ CD8+ phenotype. SLP-76 expression is then up-regulated following selection and concomitant with maturation to a CD4+ or CD8+ phenotype. In the periphery, SLP-76 is expressed in T lymphocytes with no detectable expression in the B cell compartment. Exposure to the superantigen staphylococcal enterotoxin B augments SLP-76 expression in the reactive T cell subset. Furthermore, in vitro stimulation with TCR-specific Abs augments the existing levels of SLP-76. These data reveal that SLP-76 expression is coordinately regulated with surface expression of a pre-TCR or mature TCR complex during thymocyte development and that TCR ligation elicits signals that result in increased expression of SLP-76.

Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development.

The leukocyte-specific adapter molecule SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kilodaltons) is rapidly phosphorylated on tyrosine residues after receptor ligation in several hematopoietically derived cell types. Mice made deficient for SLP-76 expression contained no peripheral T cells as a result of an early block in thymopoiesis. Macrophage and natural killer cell compartments were intact in SLP-76-deficient mice, despite SLP-76 expression in these lineages in wild-type mice. Thus, the SLP-76 adapter protein is required for normal thymocyte development and plays a crucial role in translating signals mediated by pre-T cell receptors into distal biochemical events.

Adaptor proteins in lymphocyte antigen-receptor signaling.

Adaptor molecules, proteins that possess no intrinsic enzymatic function, but which mediate protein-protein interactions, have a critical role in integrating signal transduction pathways following engagement of cell-surface receptors. Several newly described adaptor molecules have been shown to serve important functions in the regulation of signaling events initiated by lymphocyte antigen receptors. Understanding how these adaptor proteins function to modulate signaling cascades will provide important insights into the complex biology of lymphocyte activation.

Abnormal regulation of the IL-2 promoter in lpr CD4-CD8- T lymphocytes results in constitutive expression of a novel nuclear factor of activated T cells-binding factor.

The inert quality of MRL-Ipr/Ipr (Ipr) peripheral CD4-CD8- (CD4-8-) T cells manifests primarily as an inability to proliferate or produce IL-2 in response to TCR or mitogenic stimulation. Yet these same cells do initiate early TCR-mediated signaling events, such as generation of inositol phosphates and increased intracellular calcium. They also display constitutively high levels of p59fyn and CD3 zeta tyrosine phosphorylation. The generation of second messengers in T cells normally leads to downstream signaling that results in transcriptional activation of the IL-2 gene. We, therefore, compared the activation state of the IL-2 gene promoter region in freshly isolated and stimulated Ipr CD4-8- T cells with that of normal T lymphocytes. Levels of the octamer, NF-kappa B (p50-p65 heterodimer), and AP-1 transcriptional factors are constitutively elevated in freshly isolated Ipr CD4-8- T cells, consistent with the activated phenotype of these cells. Upon stimulation with mitogens, formation of the transactivating complex, nuclear factor of activated T cells (NF-AT), occurs with normal kinetics in Ipr CD4-8- T cells. Yet, the levels of the activating NF-AT complex never reach those observed in similarly stimulated normal T cells. Furthermore, nuclear extracts from Ipr CD4-8- T cells display high levels of a novel specific binding activity at the NF-AT site, which is present at much lower levels in freshly isolated normal T lymphocytes. Upon mitogenic stimulation, the binding activity of the novel NF-AT-binding factor is rapidly down-regulated in normal T cells, but persists at high levels in Ipr CD4-8- T cells. These two abnormalities at the NF-AT site provide a potential mechanism to account for the defect in IL-2 production from Ipr CD4-8- T cells.

Nifedipine (Procardia XL) as a cause of false-positive results on barium enema study.

There are a number of potential causes for false-positive results on a barium enema study. We report three cases in which patients had to have colonoscopy because a nifedipine slow-release tablet (Procardia XL) was misinterpreted as a colonic polyp on a barium enema study. The pill did not move with abdominal compression, further suggesting that it was a polyp. Patients in whom similar lesions are identified during barium enema examination should be questioned as to whether they are taking these medications.

Reversal of hyporesponsiveness in lpr CD4-CD8- T cells is achieved by induction of cell cycling and normalization of CD2 and p59fyn expression.

T cells freshly isolated from the peripheral lymph nodes of autoimmune MRL lpr/lpr (lpr) mice contain a large proportion of functionally non-mature T cell receptor (TcR)-alpha beta+CD3+CD2-CD4-CD8- T cells displaying the B cell isoform of CD45, B220. These cells are hyporesponsive as defined by minimal interleukin-2 (IL-2) production and proliferation in response to stimulation. However, increased levels of inositol phosphates and a rapid mobilization of Ca2+ do occur upon stimulation of the TcR/CD3 complex. Furthermore, lpr CD4-CD8-T cells contain high levels of transcripts for the src-family tyrosine kinase p59fyn, and express a constitutively tyrosine-phosphorylated CD3-zeta chain. These features bear a certain resemblance to anergized T cells. These similarities are extended to show that culturing of lpr CD4-CD8- T cells in the presence of IL-2 in combination with phorbol 12-myristate 13-acetate and ionomycin initiates cell cycling and results in the gain of function; re-stimulation now yields IL-2-dependent proliferation in the absence of exogenous IL-2. In parallel with this gain in function, the population of cells obtained after 1 week in culture retains the TcR-alpha beta + CD4-CD8- phenotype, yet displays increased levels of CD2, decreased surface B220, and normal amounts of p59fyn-specific transcripts. These findings show that cell cycling is associated with the recovery of functional capabilities by lprCD4-CD8-T cells and is closely allied with surface CD2 expression. Thus, the hyporesponsiveness of lpr T cells is not a fixed state.

Co-stimulation via CD28 induces activation of a refractory subset of MRL-lpr/lpr T lymphocytes.

Peripheral lymphoid tissues of lpr mice contain a large proportion of TCR alpha beta/CD3+CD4-CD8- T cells that lack surface CD2 and express the B cell isoform of CD45, B220. This subset of T cells does not proliferate or produce IL-2 in response to mitogenic signals or TCR-CD3 ligation. At the same time, these abnormal T cells display several characteristics of an activated phenotype. Collectively, these properties of lpr CD4-CD8- T cells have functional parallels with anergic T cells. A critical co-stimulatory molecule implicated in the prevention of or recovery from anergy is CD28, which binds the ligand BB1/B7 on certain accessory cells. lpr CD4-CD8- T cells express normal levels of CD28 which is capable of transducing a strong proliferative signal to these cells in co-stimulation with mitogens. However, proliferation of lpr CD4-CD8- T cells in response to CD28 co-stimulation does not reach the levels observed in normal T cells stimulated under similar conditions. Stimulation with anti-CD28 mAb in conjunction with phorbol myristate acetate and ionomycin promotes cell cycling in the CD2- subset of CD4-CD8- T cells, and results in a slight induction of CD2 levels during the course of the culture period. However, the majority of cells obtained at the end of the culture period remain TCR alpha beta+ CD4-CD8-, CD2low/- and B220high, similar to freshly isolated CD4-CD8- lpr T cells. In contrast, if IL-2 is included in the cultures, a strong shift toward a CD2+ phenotype is observed by a majority of the lpr T cells. Upon repeat stimulation, these lpr CD4-CD8- T cells can now proliferate in an IL-2-dependent manner when stimulated with only anti-CD3 mAb or mitogens, in the absence of exogenous IL-2 or anti-CD28 mAb. These data show that the hyporesponsiveness of lpr CD4-CD8- T cells does not result from a lack of CD28 expression, that it is not a fixed state, and that it can be reversed by the induction of cell cycling in the presence of IL-2. These observations extend the parallels between lpr CD4-CD8- T cells and anergic T cells.

Angiographic diagnosis of gastric volvulus with report of a complication following left gastric artery embolization.

Gastric volvulus is a rare cause of upper gastrointestinal (UGI) tract obstruction and may present as acute UGI hemorrhage. The angiographic findings of gastric volvulus are discussed and a report of a complication of embolization of the left gastric artery in unsuspected mesenteroaxial stomach volvulus is given.

Radiographic evaluation of the free jejunal graft.

Jejunal autotransplantation is an increasingly popular method of reconstructive surgery for the upper aerodigestive tract following laryngopharyngectomy. From 1977 to 1985, 55 free jejunal grafts were performed on 49 patients. Thirty patients (61%) experienced reconstructive complications including graft failure in 6, fistula in 18, and strictures in 10. Postoperative dysphagia occurred in 26 patients (53%). The cause of the dysphagia is often complex and related to a number of factors that are discussed. While jejunal autotransplantation is successful in selected patients, complications are not infrequent. The radiologist plays an important role in the evaluation and follow-up of these patients.

Percutaneous biopsy of pancreatic masses.

Forty-five percutaneous needle biopsies of pancreatic masses were performed in 41 patients in whom there was clinical or radiological concern for the diagnosis of cancer. Computed tomography (CT) was the imaging technique used in 41 of the 45 biopsies. An overall sensitivity of 85%, specificity of 100%, and accuracy rate of 88% were achieved, with serious complications in three of the 45 attempts (6.7%). These figures show that percutaneous pancreatic biopsies have the potential for greater accuracy than has been previously reported and are relatively safe when compared with surgery.

Retrocardiac densities due to paraesophageal varices: roentgenographic detection.

We have described a patient with portal hypertension and retrocardiac paramediastinal density on upright PA chest x-ray film. Splenoportograms showed this density to be paraesophageal collateral veins.

Prevalence of diverticular disease, hiatus hernia, and pelvic phleboliths in black and white Americans.

Phleboliths, and especially diverticular disease and hiatus hernia, are rarer in developing countries than in economically more developed communities, but all three conditions were as common in Black as in White Americans. This finding suggests that they are due to environmental rather than to genetic causes. A deficient intake of dietary fibre may be the common factor predisposing to these three conditions.