Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Rubella seroprevalence using residual samples from the South African measles surveillance program: a cross-sectional analytic study.

Introduction: South Africa is yet to introduce rubella-containing vaccines (RCV) into its routine immunization schedule. Selecting the target population when introducing RCV should take into account the ages of susceptible individuals in the population. We aimed to determine the seroprevalence of antibodies to rubella and characterize immunity gaps among individuals of all ages in South Africa. Methods: We tested for rubella immunoglobulin G (IgG) antibodies with a commercial enzyme-linked immunosorbent assay. We used residual samples collected from 2016 through 2018 as part of the national measles surveillance program. We only tested samples that were negative for measles and rubella immunoglobulin M (IgM) and explored the association between rubella susceptibility (IgG negative) and predictor variables (year of sample collection, age, sex, and province of residence) using logistic regression analysis. Results: We obtained results for 6057 records. Rubella susceptibility was highest among Individuals aged zero to 11 months (81.9%), followed by children 1 to 5 years old (71.5%), 6 to 10 y old (40.9%) and 11 to 15 y old (31.25) while the smallest proportion of susceptible individuals was among those 16 to 49 y old (19.9%). Females were less likely to be susceptible to rubella compared to males (OR = 0.79 (95%CI: 0.71-0.87), P < .001) in unadjusted analysis but this effect was not observed after adjusting for age and province. In multivariable logistic regression, age (OR = 6.24 (4.52-8.63), P < .001) and province of residence (OR = 0.97 (95%CI: 0.95-0.99), P = .01) were associated with rubella susceptibility. Conclusion: In the absence of rubella vaccination in the Expanded Program on Immunization in South Africa, the bulk of individuals susceptible to rubella are children under 16 y old. About 20% of individuals 16 to 49 y old are susceptible to rubella. This susceptibility gap must be born in mind during RCV introduction.

A lymphoma plasma membrane-associated protein with ankyrin-like properties.

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation.

Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1-like protein.

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin-binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.

Thrombospondin stimulates motility of human neutrophils.

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)-mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20-70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.

Characterization and cytoskeletal association of a major cell surface glycoprotein, GP 140, in human neutrophils.

The binding of specific ligands to neutrophil cell surface receptors and the association of these receptors with the cytoskeleton may represent an essential step in activation. To identify surface proteins that are linked to the cytoskeleton during activation, neutrophil 125I-surface labeled plasma membranes were extracted with Triton X-100, and the soluble and insoluble (cytoskeleton) fractions analyzed by SDS-PAGE and autoradiography. The major cell surface proteins recruited to the cytoskeleton after activation with Con A, FMLP, zymosan-activated serum, or immune complexes possessed a relative molecular mass in the range of 80 to 13 kD. In addition to these proteins, WGA stimulates the recruitment of a 140-kD protein (GP 140) to the cytoskeletal fraction. That GP 140 is a WGA-binding protein was verified by Western blotting and WGA-Sepharose affinity chromatography. The Coomassie blue staining pattern of the WGA cytoskeletal fraction revealed major protein bands at apparent molecular weights of greater than 200 (approximately 250, 240, 235), 200, 115, 82/78 (a doublet), 56, 43, 36, and 18 kD. Labeling cells with 32PO4 before WGA treatment indicated that the cytoskeletal proteins with molecular weights of 115, 82/78, and 72 kD, and a 40-kD detergent soluble protein, are phosphorylated during activation. The 78 kD cytoskeletal phosphoprotein co-migrates with the lower subunit of erythrocyte (RBC) band 4.1 and shows strong cross-reactivity with RBC anti-band 4.1 antibody. Phosphorylation of cytoskeletal proteins like 4.1 may be involved in the regulation of interactions between GP 140 and the actin-containing cytoskeleton. Unlike the C3bi receptor, GP 140 is a major surface component of unactivated PMNs, has no stoichiometrically related 95-kD subunit, and has two isoforms with pIs in the range of 6.4 to 6.6. Under conditions that result in an increased expression of the C3bi receptor (such as treatment with the Ca2+ ionophore A23187), the amount of GP 140 on the PMN cell surface appears to be significantly reduced. The interaction of GP 140 with the cytoskeleton during activation suggests that GP 140 may play an important role in neutrophil functional responses.

Motility of human carcinoma cells in response to thrombospondin: relationship to metastatic potential and thrombospondin structural domains.

The interactions of tumorigenic cells with the extracellular matrix play a critical role in the establishment of metastases. Thrombospondin (TSP) is prominent at sites of tissue injury and promotes the attachment, spreading, and motility of several cell types. We have investigated the relationship between human carcinoma cell metastatic potential and TSP-mediated cell motility by comparing highly metastatic 11B carcinoma cells with a nonmetastatic counterpart, 22B carcinoma cells. 11B cells demonstrated motility in response to soluble TSP with a maximal effect observed at 1 microM TSP. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. Monoclonal antibody C6.7, specific for the distal COOH terminus of TSP, inhibited chemotaxis by 60%. Studies with TSP fragments demonstrated that the M(r) 140,000 COOH-terminal domain (140K) supported chemotaxis to the same extent as intact TSP. The NH2-terminal heparin-binding domain was ineffective in stimulating chemotaxis. Substrate-bound TSP also elicited 11B cell motility with a maximal response at 100 nM TSP. Directionality of this response was confirmed by checkerboard analysis. Interestingly, as in chemotaxis, haptotaxis was mediated exclusively by 140K as demonstrated by TSP fragment studies and inhibition with monoclonal antibody C6.7. Therefore, 140K appeared to mediate both chemotaxis and haptotaxis. Compared with 11B cells, 22B carcinoma cells are nonmetastatic and synthesize and secrete low levels of TSP. Immunoprecipitation and Northern blot analysis confirmed that 11B cells expressed much higher levels of TSP than 22B cells. Although 22B cells are able to attach to TSP, they did not exhibit either chemotaxis or haptotaxis in response to TSP or TSP fragments. Similarly, an antisense TSP cell line responded poorly in chemotaxis assays to TSP and 140K. These data suggest that the ability of metastatic cells in vivo to establish secondary sites of proliferation may be related to their ability to migrate in response to extracellular matrix proteins such as TSP incorporated into basement membranes or interstitial matrices.

Further characterization of a fodrin-containing transmembrane complex from mouse T-lymphoma cells.

A transmembrane complex containing fodrin (an actin-binding protein) and a major surface glycoprotein (GP 180) was previously isolated from mouse T-lymphoma cells by the complementary techniques of non-ionic detergent extraction and sucrose gradient centrifugation (Bourguignon et al. (1985) J. Cell Biol. 101, 477-487). The analysis of this complex has been extended to verify the structural association and further define the interaction between fodrin and GP 180. The association between fodrin and GP 180 has been confirmed by the following evidence: co-sedimentation of fodrin and GP 180 in a single peak on a sucrose gradient with a sedimentation coefficient of 20 S; a constant ratio of fodrin and GP 180 across the 20 S peak; the specific co-precipitation of GP 180 with fodrin from the 20 S peak using anti-fodrin antibody; and the colocalization of fodrin and GP 180 from the 20 S peak on actin filaments using an immuno-electron microscopic technique. Furthermore, this fodrin-GP 180 complex can be readily dissociated and reassembled in the presence and absence of 0.6 M NaCl, respectively. The fact that this fodrin-GP 180 complex displays actin-binding ability indicates that this transmembrane complex may play an important role in the linking event between receptors and the cytoskeleton during lymphocyte patching and capping.

Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis-(O-aminophenoxy)-ethanol-N,N;N'-tetraacetic acid (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA-treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn-1,2-didecanoylglycerol to the BAPTA-treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs.

Transforming growth factor-beta1 stimulates degranulation and oxidant release by adherent human neutrophils.

The signal transduction pathways that are activated by cytokines and growth factors binding to their receptors on human neutrophils (PMN) are poorly understood. When PMN in suspension encounter many of these agonists they are not activated, but rather are primed for subsequent activation. We and others reported that when PMN are plated onto fibrinogen and stimulated with cytokines or with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) they respond by releasing hydrogen peroxide (H202) and the specific granule component lactoferrin. Transforming growth factor-beta1 (TGF-beta1) is released by many cells including PMN. It has been reported that TGF-beta1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF-beta1 would activate PMN to release H202 when they were adherent to fibrinogen, a response mediated by beta2++integrin receptors. In this study, we determined whether TGF-beta1 stimulated H202 and lactoferrin release by PMN adherent to fibrinogen. TGF-beta1 stimulated H202 and lactoferrin release from adherent PMN in a concentration-dependent manner, with effects seen in the range of 0.1 to 100 pg/mL. Both H202 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H202 and lactoferrin release. Ethanol (200 mM) blocked both H202 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso-[3H]phosphatidylcholine, we observed that TGF-beta1 treatment caused an increase in [3H]phosphatidate. Propranolol (150 microM), an inhibitor of phosphatidate phosphohydrolase, blocked both H202 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF-beta1. Overall, these results are similar to those reported for fMLP activation of adherent PMN and suggest that a common pathway is involved in both chemoattractant and cytokine activation.

Potent reversible inhibitors of the protein tyrosine phosphatase CD45.

The cytosolic portion of CD45, a major transmembrane glycoprotein found on nucleated hematopoietic cells, contains protein tyrosine phosphatase activity and is critical for T-cell receptor-mediated T-cell activation. CD45 inhibitors could have utility in the treatment of autoimmune disorders and organ graft rejection. A number of 9,10-phenanthrenediones were identified that reversibly inhibited CD45-mediated p-nitrophenyl phosphate (pNPP) hydrolysis. Chemistry efforts around the 9,10-phenanthrenedione core led to the most potent inhibitors known to date. In a functional assay, the compounds were also potent inhibitors of T-cell receptor-mediated proliferation, with activities in the low micromolar range paralleling their enzyme inhibition. It was also discovered that the nature of modification to the phenanthrenedione pharmacophore could affect selectivity for CD45 over PTP1B (protein tyrosine phosphatase 1B) or vice versa.

Interaction of human neutrophils and HL-60 cells with the extracellular matrix.

The accumulation of polymorphonuclear leukocytes (PMNs) at sites of inflammation or injury is generally attributed to the presence of chemoattractants and agents that increase PMN adherence. Extracellular matrix (ECM) proteins released at these sites may promote or modulate PMN adhesion, motility, oxidant generation, degranulation, and phagocytosis, but their role in these processes is not well defined. Of particular interest are thrombospondin (TSP), a 450 kD ECM protein released by activated platelets, and vitronectin (VN), a major constituent of plasma. Low concentrations of soluble TSP prime for both N-formyl-methionyl-leucyl-phenylalanine (FMLP)-mediated O2- generation and chemotaxis, whereas VN suppresses FMLP-mediated O2- generation but primes for FMLP-mediated chemotaxis. TSP alone, at high concentrations, stimulates chemotaxis of PMNs, whereas VN, at the same concentrations, fails to stimulate chemotaxis. In contrast to soluble ECM proteins, substrate bound TSP, laminin, and VN promote PMN adhesion and random migration. As functional studies suggest, unactivated PMNs express receptors for both TSP and VN, and both TSP and VN receptor expression increases substantially following PMN activation. PMN-like HL-60 cells interact similarly with ECM proteins, and thus provide an important model for studying the expression of ECM receptors and the acquisition of ECM-mediated functional responses during blood cell differentiation. The ability of PMNs to interact with ECM proteins and modulate ECM protein receptors suggests that these proteins, alone or in synergy with chemotactic peptides, play an important role in regulating PMN diapedesis.

Potential electronic transition chemical laser: parametric evaluation.

Through the utilization of a generalized computer code that calculates the minimum reactive branching ratio required for a species to display optical gain, a parametric study was performed to ascertain which properties of the electronic potential curves of a heteronuclear diatomic molecule have the greatest effect on the suitability of that molecule as a potential electronic transition laser. The results of this study demonstrate that diatomic systems separate into different classes when the question of the minimum required reactive branching ratio is confronted. This separation of molecular systems was translated through the use of compiled molecular constants into a determination of which vibrational-level distribution class is most favorable for specific heteronuclear diatomic molecules to display optical gain; potential laser candidates were pointed out. In addition, a generalized gain equation for electronic transitions heteronuclear diatomic molecules that takes into account both rotational and vibrati nal partitioning was derived.

Thrombospondin promotes chemotaxis and haptotaxis of human peripheral blood monocytes.

The presence of the extracellular matrix protein thrombospondin (TSP) at sites of tissue injury or inflammation may promote monocyte migration to these sites and play a central role in their eventual differentiation into tissue macrophages. Previously, we have shown that TSP promotes neutrophil adhesion and migration, and primes for oxidant generation. To examine the effect of TSP on monocyte motility, we conducted chemotaxis assays in modified Boyden chambers. TSP was chemotactic for monocytes, with a maximal response at 200 to 500 nM TSP. Checkerboard analysis confirmed that migration was directional. mAb C6.7, against the distal COOH terminus of TSP, inhibited chemotaxis, demonstrating specificity and indicating that the chemotactic activity resides in the COOH terminus. Consistent with the mAb data, the COOH-terminal 140-kDa proteolytic fragment of TSP was chemotactic for monocytes, whereas the NH2-terminal heparin-binding domain was inactive. A synthetic peptide containing the sequence CSVT, derived from the type I repeats of TSP, was also chemotactic. Thus, two different sites on the COOH terminus of TSP are capable of stimulating monocyte chemotaxis. Pertussis toxin, but not cholera toxin, completely inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. TSP bound to polycarbonate filters stimulated monocyte haptotaxis, with a maximal response at 4 pmol. The directional nature of this motility was confirmed by checkerboard analysis. Monocyte haptotaxis was inhibited by two different mAbs recognizing distinct sites on the COOH terminus. As with chemotaxis, the 140-kDa fragment, but not the heparin-binding domain, contained the haptotactic activity. The CSVT-containing synthetic peptide also promoted monocyte haptotaxis. But, in contrast to chemotaxis, neither pertussis toxin nor cholera toxin inhibited TSP-mediated haptotaxis, suggesting the involvement of a different signal transduction pathway. mAbs against GPIV, beta 1, beta 3, or alpha v integrins did not affect monocyte chemotaxis or haptotaxis, ruling out the involvement of these receptors. These results indicate that TSP is likely to play an important role in monocyte recruitment to an inflammatory or injury site.

Exocytosis of a subpopulation of specific granules coincides with H2O2 production in adherent human neutrophils.

Unlike polymorphonuclear leukocytes (PMN) that are activated in suspension, PMN plated onto extracellular matrix (ECM) proteins exhibited a prolonged delay in the onset of the respiratory burst in response to PMA, FMLP, A23187, or GM-CSF. The present study was focused on examining the events leading to H2O2 release in adherent PMN. The time course of stimulated H2O2 release from PMN plated on fibrinogen (FG) kinetically paralleled cell spreading and lactoferrin release. In contrast, the release of another specific granule component, vitamin B12-binding protein, preceded H2O2 generation by at least 20 min, suggesting the differential mobilization of subpopulations of specific granules. FMLP-stimulated PMN from a patient with leukocyte adhesion deficiency (LAD), lacking CD11/CD18 integrins and unable to adhere to FG, attached and spread on thrombospondin (TSP) and generated substantial amounts of H2O2. However, PMN from a second LAD patient, able to attach but not spread on TSP, failed to generate oxidant. These data indicated that PMN spreading might be a prerequisite for H2O2 generation and that CD11/CD18 integrins are likely not the only surface receptors involved in this response. Exposing PMN to the intracellular Ca2+ chelators MAPTAM or BAPTA significantly reduced H2O2 generation in response to the receptor-mediated agonists, FMLP or GM-CSF, but did not affect PMA-stimulated H2O2 generation. Under conditions that resulted in reduced H2O2 generation (i.e., MAPTAM or BAPTA plus FMLP or GM-CSF), lactoferrin, but not vitamin B12-binding protein, release was also reduced, suggesting a link between the secretion of lactoferrin-containing specific granules and H2O2 production. Since neither MAPTAM or BAPTA blocked PMN spreading, it appeared that cell spreading alone was not sufficient for H2O2 production. Thus, the major requirement for H2O2 generation appeared to be the exocytosis of a distinct population of lactoferrin-containing specific granules. In support of this observation, PMN-like HL-60 cells, which lack specific granules, attached and spread on FG but failed to release H2O2. Thus, we postulate that the delivery of cytochrome b from lactoferrin-containing specific granules to the plasma membrane during activation contributes to oxidant production in PMN adherent to ECM proteins.

Neutrophil thrombospondin receptors are linked to GTP-binding proteins.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP-binding proteins (G-proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP-mediated motility. Both PT and CT inhibited TSP-mediated chemotaxis and substrate-associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 microM) nor 8-bromo-cAMP (300 microM) significantly affected TSP-mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 +/- 0.3 to 14.2 +/- 0.1 pmol/10(6) cells). Although we could not test the PT sensitivity of TSP priming for FMLP-mediated chemotaxis (as PT inhibits FMLP-mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP-dependent priming of FMLP-mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G-proteins was obtained by examining the effect of TSP on alpha-subunit ADP-ribosylation, GTPase activity, and GTP gamma S binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT-dependent ribosylation of Ci alpha 2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G-proteins. However, TSP did not block the CT-dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of Gi alpha 2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 +/- 2.7% of control values). In addition, GTP gamma S binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 +/- 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G-proteins, a subpopulation of which also associates with FMLP receptors.

Thrombospondin promotes both chemotaxis and haptotaxis in neutrophil-like HL-60 cells.

Differentiation of the human promyelocytic leukemia cell line HL-60 to polymorphonuclear leukocyte (PMN)-like cells with DMSO provides an important model for studying the acquisition of PMN functional responses that accompany differentiation. We showed previously that the extracellular matrix (ECM) protein thrombospondin (TSP) binds to PMN surface receptors and promotes adhesion and motility. Undifferentiated HL-60 cells did not adhere and were not motile in response to TSP, whereas cells differentiated toward PMN-like cells demonstrated both TSP-mediated adhesion and chemotaxis, with chemotaxis evident by day 2 of induction. With differentiation, a maximal response was obtained with 100 to 300 nM TSP, 10-fold lower than required for maximal PMN chemotaxis. Checkerboard analysis confirmed the directional nature of motility. mAb recognizing different domains of TSP inhibited chemotaxis, suggesting the involvement of multiple sites on TSP. Although both the NH2-terminal heparin-binding domain (HBD) and 140 kDa COOH-terminal fragment supported chemotaxis in PMN-like cells, neither fragment was as potent as intact TSP. Both pertussis and cholera toxin inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. The toxin effects did not indirectly result from elevated cAMP levels because high concentrations of either 8-bromo-cAMP or dibutyryl cAMP did not inhibit chemotaxis. TSP bound to nitrocellulose filters induced the directed migration (haptotaxis) of PMN-like cells rather than the random motility observed with PMN. Haptotaxis was stimulated by either the HBD or 140-kDa fragment and was inhibited by mAb against these two domains. Haptotaxis rather than random migration was confirmed by checkerboard analysis. Our results demonstrate that PMN-like HL-60 cells respond differently to TSP than human peripheral blood PMN. These differences may reflect 1) an aberration in HL-60 differentiation reflecting their leukemic phenotype 2) differentiation of HL-60 cells to a cell type characteristic of "activated" PMN.

Syk activation is required for spreading and H2O2 release in adherent human neutrophils.

Chemoattractant-stimulated polymorphonuclear leukocytes (PMNs) that are adherent to extracellular matrix proteins exhibit a massive, sustained respiratory burst that requires cell spreading. However, the signaling pathways culminating in PMN spreading are not well characterized. Studies showing that protein tyrosine phosphorylation increases with PMN spreading suggest that phosphorylation is critical for this process. In the present study, we observed increased tyrosine phosphorylation of both focal adhesion kinase and Syk in FMLP-activated PMNs that had been plated onto fibrinogen; an increase in Syk activity, but not focal adhesion kinase activity, was apparent. The time course of Syk phosphorylation correlated with the initiation of cell spreading and H2O2 release. Pretreatment of PMNs with piceatannol, a Syk-selective inhibitor, blocked Syk activity, cell spreading, and H2O2 release, indicating that Syk activity was required for the activation of adherent PMNs. Paxillin is a cytoskeletally associated protein that is also tyrosine phosphorylated during PMN spreading and H2O2 release. Paxillin phosphorylation is kinetically slower than Syk phosphorylation and is inhibited with piceatannol, suggesting that paxillin is a substrate for Syk. An analysis of Syk immunoprecipitates indicated that Syk and paxillin associate during PMN spreading. This interaction is not mediated by the src kinases Lyn and Fgr, since neither kinase coprecipitated with Syk. Syk from FMLP-activated, adherent PMNs phosphorylated paxillin-glutathione S-transferase, suggesting that paxillin is a substrate for Syk in vivo. These results indicate that PMN spreading and H2O2 release require a Syk-dependent signaling pathway leading to paxillin phosphorylation.