Co-stimulation and counter-stimulation: lipid raft clustering controls TCR signaling and functional outcomes.

T cell receptor (TCR) antigen recognition induces the formation of a specialized 'immunological synapse' at the T cell : antigen presenting cell (APC) junction. This junction is generated by the recruitment and exclusion of particular proteins from the contact area and is required for T cell activation. We and others have hypothesized that lipid raft/non-raft partitioning provides a molecular basis for protein sorting which organizes the TCR, co-stimulators, signal transducers and the actin cytoskeleton at the T cell : APC interface. Here we discuss the emerging paradigm that co-stimulators induce the directional transport and clustering of lipid rafts at the T cell : APC interface, thus generating platform(s) specialized for processive and sustained TCR signal transduction and T cell activation. We also discuss recent data implicating the involvement of 'counter-stimulators' and other negative regulators which prevent optimal raft clustering at the TCR contact site and, thus, facilitate T cell inactivation and tolerance induction.

A molecular framework for two-step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events.

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.

Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction.

Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-gamma production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-zeta and the generation of inhibitory pp21zeta. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell "counterstimulator" to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21zeta-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC.

C-C chemokine receptor 3 antagonism by the beta-chemokine macrophage inflammatory protein 4, a property strongly enhanced by an amino-terminal alanine-methionine swap.

Allergic reactions are characterized by the infiltration of tissues by activated eosinophils, Th2 lymphocytes, and basophils. The beta-chemokine receptor CCR3, which recognizes the ligands eotaxin, eotaxin-2, monocyte chemotactic protein (MCP) 3, MCP4, and RANTES, plays a central role in this process, and antagonists to this receptor could have potential therapeutic use in the treatment of allergy. We describe here a potent and specific CCR3 antagonist, called Met-chemokine beta 7 (Ckbeta7), that prevents signaling through this receptor and, at concentrations as low as 1 nM, can block eosinophil chemotaxis induced by the most potent CCR3 ligands. Met-Ckbeta7 is a more potent CCR3 antagonist than Met- and aminooxypentane (AOP)-RANTES and, unlike these proteins, exhibits no partial agonist activity and is highly specific for CCR3. Thus, this antagonist may be of use in ameliorating leukocyte infiltration associated with allergic inflammation. Met-Ckbeta7 is a modified form of the beta-chemokine macrophage inflammatory protein (MIP) 4 (alternatively called pulmonary and activation-regulated chemokine (PARC), alternative macrophage activation-associated C-C chemokine (AMAC) 1, or dendritic cell-derived C-C chemokine (DCCK) 1). Surprisingly, the unmodified MIP4 protein, which is known to act as a T cell chemoattractant, also exhibits this CCR3 antagonistic activity, although to a lesser extent than Met-Ckbeta7, but to a level that may be of physiological relevance. MIP4 may therefore use chemokine receptor agonism and antagonism to control leukocyte movement in vivo. The enhanced activity of Met-Ckbeta7 is due to the alteration of the extreme N-terminal residue from an alanine to a methionine.

The Lck SH2 phosphotyrosine binding site is critical for efficient TCR-induced processive tyrosine phosphorylation of the zeta-chain and IL-2 production.

The lymphocyte-specific tyrosine kinase Lck is essential for TCR-mediated signal transduction. This is in part due to its enzymatic activity as a tyrosine kinase responsible for TCR-induced tyrosine phosphorylation of zeta and CD3 receptor subunits. In addition to its catalytic domain, the Lck protein contains SH3 and SH2 domains capable of associating with other signaling molecules. It has been proposed that phosphotyrosine binding by the Lck SH2 domain may enhance substrate tyrosine phosphorylation by facilitating the processive phosphorylation of multiple sites within the TCR complex. Alternatively or additionally, it may function in adapter activity for facilitating required protein-protein interactions. Previous experiments demonstrate that overexpression of a constitutively activated form of Lck (F505) in the BI-141 T cell hybridoma leads to the Lck kinase activity-dependent enhancement of TCR-mediated signals. Here we demonstrate that mutation of amino acids important for SH2 phosphotyrosine binding significantly compromises the ability of F505 to enhance TCR-mediated protein tyrosine phosphorylation and Ag-induced IL-2 production in BI-141. Examination of the effects of TCR-regulated phosphorylation of the Lck substrate zeta provides in vivo evidence for a role for the Lck SH2 domain in the processive phosphorylation of a multiply phosphorylated substrate.

Specific domains of fibronectin mediate adhesion and migration of early murine erythroid progenitors.

The binding of late stage erythroid cells to fibronectin (FN) has been well characterized and is believed to be critical for the terminal stages of erythroid differentiation, but the adhesive properties of more primitive murine erythroid progenitors and the role of these interactions during earlier stages of erythropoiesis has not been determined. Using chymotryptic fragments and inhibitory probes, we have tested the ability of each of the major cell binding domains of FN; the RGDS sequence, the CS-1 sequence, and the carboxy-terminal heparin-binding domain (HBD), to promote adhesion of primitive burst-forming unit-erythroid (BFU-E), mature BFU-E, and colony-forming unit-erythroid (CFU-E). We found that only 10% to 15% of BFU-E bound to FN or to the RGDS sequence in contrast to 75% to 85% of CFU-E. Approximately 50% to 70% of BFU-E and 60% to 80% of CFU-E bound to the carboxy-terminal HBD and to the CS-1 sequence. The binding of BFU-E and CFU-E to the RGDS and CS-1 sites was blocked by beta1 integrin antibodies. These results suggest that binding to FN determinants is developmentally regulated during early erythroid differentiation. Erythroid progenitor migration within the bone marrow is thought to be important for the eventual release of reticulocytes into the circulation. A correlation between FN binding and the migratory capacity of erythroid cells has been suggested, although the ability of FN to promote migration of erythroid progenitors has not been directly measured. We measured migration of CFU-E on fragments of FN containing each cell binding region. CS-1-containing fragments, in addition to promoting adhesion of both BFU-E and CFU-E, supported the highest levels of CFU-E migration (11-fold above background). Migration was sixfold above background on intact FN and only threefold above background on RGDS-containing fragments. Fragments containing HBD alone, although they promoted adhesion of CFU-E, failed to support significant levels of migration. These results show that specific domains of FN possess distinct adhesion- and migration-promoting properties for murine erythroid progenitors. Regulation of the adhesive properties during erythroid differentiation may alter the ability of progenitors to migrate in the bone marrow and thus play an important role in normal murine erythroid differentiation.

Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors.

Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.

Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.

Direct contact between hematopoietic cells and viral packaging cell lines or other sources of stroma has been shown to increase the efficiency of retroviral-mediated gene transfer into these target cells compared with infection with viral supernatant. We have investigated the role of defined bone marrow extracellular matrix molecules (ECM) in this phenomenon. Here we report that infection of cells adhering to the carboxy-terminal 30/35-kD fragment of the fibronectin molecule (30/35 FN), which contains the alternatively spliced CS-1 cell adhesion domain, significantly increases gene transfer into hematopoietic cells. Two retroviral vectors differing in recombinant viral titer were used. Gene transfer into committed progenitor cells and long-term culture-initiating cells, an in vitro assay for human stem cells, was significantly increased when the cells were infected while adherent to 30/35 FN-coated plates compared with cells infected on BSA-coated control plates or plates coated with other bone marrow ECM molecules. Although gene transfer into committed progenitor cells and to a lesser degree into long-term culture-initiating cells was increased on intact fibronectin as well, increased gene transfer efficiency into hematopoietic cells on 30/35 FN was dependent on CS-1 sequence since infection on a similar FN fragment lacking CS-1 (42 FN) was suboptimal. 30/35 FN has previously been shown by our laboratory and other investigators to mediate adhesion of primitive murine and human hematopoietic stem cells to the hematopoietic microenvironment. Additional studies showed that neither soluble 30/35 FN nor nonspecific binding of hematopoietic cells to poly-L-lysine-coated plates had any appreciable effect on the infection efficiency of these cells. Our findings indicate that hematopoietic stem cell adhesion to specific ECM molecules alters retroviral infection efficiency. These findings should aid in the design of gene transfer protocols using hematopoietic progenitor and stem cells for somatic gene therapy.

Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions.

The self-renewal and differentiation of haematopoietic stem cells occurs in vivo and in vitro in direct contact with cells making up the haematopoietic microenvironment. In this study we used adhesive ligands and blocking antibodies to identify stromal cell-derived extracellular matrix proteins involved in promoting attachment of murine haematopoietic stem cells. Here we report that day-12 colony-forming-unit spleen (CFU-S12)5 cells and reconstituting haematopoietic stem cells attach to the C-terminal, heparin-binding fragment of fibronectin by recognizing the CS-1 peptide of the alternatively spliced non-type III connecting segment (IIICS) of human plasma fibronectin. Furthermore, CFU-S12 stem cells express the alpha 4 subunit of the VLA-4 integrin receptor, which is known to be a receptor for the CS-1 sequence, and monoclonal antibodies against the integrin alpha 4 subunit of VLA-4 block adhesion of CFU-S12 stem cells to plates coated with the C-terminal fibronectin fragment. Finally, polyclonal antibodies against the integrin beta 1 subunit of VLA-4 inhibit the formation of CFU-S12-derived spleen colonies and medullary haematopoiesis in vivo following intravenous infusion of antibody-treated bone marrow cells.

A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.

Erythroid differentiation of murine erythroleukemia (MEL) cells is far more extensive when the cells are attached to fibronectin-coated dishes than in suspension culture. Cells induced in suspension culture for 4 d become arrested at a late erythroblast stage and do not undergo enucleation. Incubation of cells in suspension beyond 4 d results in lysis. In contrast, cells induced by DMSO on fibronectin-coated dishes for 7 d differentiate into enucleating cells, reticulocytes, and erythrocytes. As determined by quantitative immunoblotting, cells induced in suspension culture accumulate approximately 33% of the amount of the major erythroid membrane protein Band 3 present in erythrocyte, whereas cells induced on fibronectin-coated dishes accumulate 80-100% of the amount present in erythrocytes. Both suspension-induced cells and cells induced on fibronectin-coated dishes accumulate approximately 90% of the amount of spectrin and ankyrin present in erythrocytes. As revealed by immunofluorescence microscopy during enucleation of MEL cells, both Band 3 and ankyrin are sequestered in the cytoplasmic fragment of the emerging reticulocyte. Enucleated and later-stage cells detach from the fibronectin matrix, due to the loss of the surface fibronectin receptor; this mimics the normal release of reticulocytes from the matrix of the bone marrow into the blood. Thus a fibronectin matrix provides a permissive microenvironment within which erythroid precursor cells reside, proliferate, migrate, and express their normal differentiation program.

Lymphoid precursor cells adhere to two different sites on fibronectin.

Several precursor lymphoid cell lines, blocked at specific stages of differentiation, adhere specifically to fibronectin in vitro. Whereas the Ba F3 cell line, which has both immunoglobulin heavy- and light-chain genes in germline configuration, interacts with the arg-gly-asp-containing cell-binding domain of fibronectin, the B-committed line PD 31, which is undergoing rearrangement of immunoglobulin light-chain genes, does not. Accordingly the Ba F3, but not the putative PD 31 surface fibronectin receptor, binds to an affinity matrix containing the 115-kD cell-binding domain of fibronectin. PD 31 cells recognize a different domain of the fibronectin molecule, which is contained within the carboxy terminal segment possessing a high-affinity binding site for heparin. A polyclonal antibody raised against the fibronectin receptor of mouse erythroleukemic cells inhibits adhesion of these lymphoid lines to fibronectin. It precipitates two major species of 140 and 70 kD from surface-radioiodinated Ba F3 cells and species of 140 and 120 kD from PD 31 cells. We propose that the two types of cells express different fibronectin receptors mediating substrate adhesion, and suggest that receptor(s) with different specificity might be expressed in the course of B cell maturation. Because we show that these adhesion properties are shared by normal bone marrow lymphoid precursors, we infer that these receptors may play a role in normal lymphopoiesis.

Sorbitol intolerance in adults. Prevalence and pathogenesis on two continents.

The prevalence and pathogenesis of sorbitol intolerance in adults have not been adequately studied. On oral administration of sorbitol (10 g), 32% of the 124 healthy adults (41 in the U.S.A., 83 in India) developed abdominal symptoms. The orocecal transit time, measured in Asian Indian volunteers by breath hydrogen (H2) analysis, was significantly shorter in those intolerant to sorbitol (71.8 +/- 36.9 min) than in those who were tolerant (109.5 +/- 47.8 min). Our results indicate that sorbitol intolerance is a potential clinical problem in a substantial number of healthy adults. A short orocecal transit time may be responsible for sorbitol intolerance in some people. We discuss the sorbitol content of common foodstuffs.

Factors affecting fasting breath hydrogen levels in healthy adults: a study in two continents.

Factors affecting fasting breath hydrogen (FBH) levels, which are known to vary in healthy individuals, have not been clearly identified. To study the effect of dietary habits on FBH levels, we tested 102 healthy adults in the United States (n = 33, group A) and India (n = 69, group C). Twenty-two Indian immigrants in the United States (group B) were also tested to study the effects of migration on FBH levels. Volunteers in groups A and B had similar FBH levels in comparison to those noted in group C, which were significantly higher. Orocecal transit time determined in these three groups supported our hypothesis that shorter transit times are associated with higher FBH levels. We recommend that the "normals" for FBH levels in population groups with different dietary habits be established for their diagnostic usefulness.

Efficacy of activated charcoal in reducing intestinal gas: a double-blind clinical trial.

Available data on the efficacy of activated charcoal in reducing lower intestinal gas and accompanying symptoms are conflicting. We conducted a double-blind clinical trial on two population groups in the United States (n = 30) and India (n = 69) known to differ in their dietary habits and ecology of gut flora. Using lactulose as the substrate, breath hydrogen levels were measured to quantify the amount of gas produced in the colon. In comparison to a placebo, activated charcoal significantly (p less than 0.05) reduced breath hydrogen levels in both the population groups. Symptoms of bloating and abdominal cramps attributable to gaseousness were also significantly reduced in both groups by activated charcoal.

Relationship of major phosphorylation reactions and MgATPase activities to ATP-dependent shape change of human erythrocyte membranes.

Human erythrocyte ghosts prepared by hemolysis and washing in hypotonic Tris are crenated by salt and divalent cations, but undergo shape change to smooth biconcave discs and stomatocytic forms when incubated with MgATP at 37 degrees C. This is normally accompanied by protein and lipid phosphorylations in which the major phosphate acceptors are the spectrin beta-chain and inositol phospholipids, respectively. The system was manipulated in several ways to demonstrate the independence of ATP-dependent shape change from the major phosphorylation reactions. Salt-extracted membranes incubated with adenosine, an inhibitor of spectrin and phosphatidylinositol kinases, underwent normal shape change despite reductions of greater than 90% in spectrin and phospholipid labeling by [gamma-32P]ATP. ATP-dependent shape change was blocked by vanadate at micromolar concentrations (half-maximal inhibition at less than 1 microM), but vanadate did not inhibit membrane autophosphorylation reactions or turnover of spectrin- or lipid-bound phosphate. Vanadate inhibited part of the ATP hydrolysis that accompanies shape change and is expressed in the presence of ouabain and EGTA. The vanadate-sensitive MgATPase activity was approximately 3 nmol Pi X min-1 X mg of protein-1. The results implicate it in ATP-dependent shape change.

The fibronectin receptor on mammalian erythroid precursor cells: characterization and developmental regulation.

The plasma membrane of murine erythro-leukemia (MEL) cells contains a 140-kD protein that binds specifically to fibronectin. A 125I-labeled 140-kD protein from surface-labeled uninduced MEL cells was specifically bound by an affinity matrix that contained the 115-kD cell binding fragment of fibronectin, and specifically eluted by a synthetic peptide that has cell attachment-promoting activity. The loss of this protein during erythroid differentiation was correlated with loss of cellular adhesion to fibronectin. Both MEL cells and reticulocytes attached to the same site on fibronectin as do fibroblasts since adhesion of erythroid cells to fibronectin was specifically blocked by a monoclonal antibody directed against the cell-binding fragment of fibronectin and by a synthetic peptide containing the Arg-Gly-Asp-Ser sequence found in the cell-binding fragment of fibronectin. Erythroid cells attached specifically to surfaces coated either with the 115-kD cell-binding fragment of fibronectin or with the synthetic peptide-albumin complex. Thus, the erythroid 140-kD protein exhibits several properties in common with those described for the fibronectin receptor of fibroblasts. We propose that loss or modification of this protein at the cell surface is responsible for the loss of cellular adhesion to fibronectin during erythroid differentiation.

Comparison of bentiromide test and rice flour breath hydrogen test in the detection of exocrine pancreatic insufficiency.

Bentiromide test (BT) has been recently approved in the United States for screening patients with chronic pancreatitis for exocrine insufficiency. A few reports have suggested that the rice flour breath hydrogen test (RFBHT)--i.e., breath hydrogen analysis after rice flour ingestion--may also be useful in diagnosing exocrine pancreatic insufficiency. We conducted this study to compare the diagnostic value of these two tests in chronic alcoholic (n = 14) and nutritional or tropical (n = 6) pancreatitis. False-positive results were not noted with either of these two tests in 12 healthy volunteers. BT was positive in 28.6% of patients with chronic alcoholic pancreatitis and in 16.7% of patients with tropical pancreatitis. In comparison, RFBHT was almost twice as sensitive as BT in detecting insufficiency in patients with alcoholic pancreatitis (50 vs. 28.6%) and four times as sensitive in patients with tropical pancreatitis (66.7 vs. 16.7%). Only one patient in our study had a positive BT but a negative RFBHT. We recommend RFBHT as a simple, safe, and inexpensive test in screening patients for exocrine pancreatic insufficiency.

Mammalian reticulocytes lose adhesion to fibronectin during maturation to erythrocytes.

We describe three situations in which a large fraction of circulating red blood cells attach tightly and specifically to fibronectin: (i) rabbits made anemic by repeated bleeding, (ii) patients with hemolytic anemia and functional asplenia and splenectomized normal humans, and (iii) splenectomized mice. Upon induction of anemia in rabbits, the proportion of circulating red blood cells capable of specifically attaching to fibronectin-coated plastic increased in parallel with the number of reticulocytes. Fibronectin-adherent red cells were barely detectable when the rabbit had recovered from the anemia. Attachment of reticulocytes to fibronectin was specific; cells did not attach to dishes coated with albumin, laminin, or collagen. None of these proteins promoted the attachment of normal erythrocytes. About 75% of the erythrocytes from splenectomized mice (but not control mice) also attached specifically to fibronectin 40 days after surgery. The effect of splenectomy was incomplete and transient; adherent cells were not detectable 8 weeks after splenectomy. As judged by labeling studies with [35S]methionine, newly emergent reticulocytes preferentially attached to fibronectin. We suggest that about half of the reticulocytes in erythropoietically unstressed mice lose their ability to attach to fibronectin, possibly due to loss of fibronectin-adhesive components, during passage through the spleen. The others lose their ability to interact with fibronectin before release, in the bone marrow, or in some extrasplenic site.