Effects of CXC chemokines on neutrophil activation and sequestration in hepatic vasculature.

The initiating step of neutrophil-induced cytotoxicity in the liver is the recruitment of these phagocytes into sinusoids. The aim of our study was to compare the efficacy of systemic exposure with individual inflammatory mediators on neutrophil activation and sequestration in the hepatic vasculature of C3Heb/FeJ mice as assessed by flow cytometry and histochemistry, respectively. The CXC chemokine macrophage inflammatory protein-2 (MIP-2; 20 microg/kg) induced a time-dependent upregulation of Mac-1 (318% at 4 h) and shedding of L-selectin (41% at 4 h). MIP-2 treatment caused a temporary increase of sinusoidal neutrophil accumulation at 0.5 h [97 +/- 6 polymorphonuclear leukocytes (PMN)/50 high-power fields (HPF)], which declined to baseline (8 +/- 2) at 4 h. The CXC chemokine KC was largely ineffective in activating neutrophils or recruiting them into the liver. Cytokines (tumor necrosis factor-alpha and interleukin-1alpha) and cobra venom factor substantially increased Mac-1 expression and L-selectin shedding on neutrophils and caused stable sinusoidal neutrophil accumulation (170-220 PMN/50 HPF). Only cytokines induced venular neutrophil margination. Thus CXC chemokines in circulation are less effective than cytokines or complement in activation of neutrophils and their recruitment into the hepatic vasculature in vivo.

Differential protection with inhibitors of caspase-8 and caspase-3 in murine models of tumor necrosis factor and Fas receptor-mediated hepatocellular apoptosis.

Excessive apoptosis has been implicated in a number of acute and chronic human diseases. The activation of caspases has been shown to be critical for the apoptotic process. The objective of this investigation was to evaluate the beneficial effects and mechanism of action of the caspase-8 inhibitor IETD-CHO and the caspase-3 inhibitor DEVD-CHO against tumor necrosis factor (TNF)-induced hepatocellular apoptosis in vivo and compare these results to effects of the same inhibitors against Fas-induced apoptosis. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine/100 microg/kg endotoxin induced parenchymal apoptosis (indicated by caspase-3 activation and morphology) and severe liver injury (indicated by the increase in plasma alanine aminotransferase activities and histology) at 7 h. Treatment with IETD-CHO or DEVD-CHO (10 mg/kg at 3, 4.5, and 5.5 h) significantly attenuated caspase-3 activation and liver injury. Western analysis showed that DEVD-CHO had no effect while IETD-CHO substantially reduced procaspase-3 and procaspase-9 processing. On the other hand, caspase-3 activation and liver injury by the anti-Fas antibody Jo-2 was completely prevented by a single dose of DEVD-CHO and, as previously shown, by IETD-CHO at 90 min. Both inhibitors prevented procaspase-3 and procaspase-9 processing. Thus, there are fundamental differences in the efficacy of caspase inhibitors in these two models. We conclude that Fas may rely exclusively on caspase-8 activation and mitochondria to activate caspase-3, which can process more procaspase-8 and thus propagate the amplification of the apoptotic signal. TNF can activate a similar signaling pathway. However, alternative signaling mechanisms seem to exist, which can compensate if the main pathway is blocked.

Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: role of mitochondrial oxidant stress.

Peroxynitrite may be involved in acetaminophen-induced liver damage. However, it is unclear if peroxynitrite is generated in hepatocytes or in the vasculature. To address this question, we treated C3Heb/FeJ mice with 300 mg/kg acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite formation. Vascular nitrotyrosine staining was evident before liver injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine staining between 2 and 6 h after acetaminophen. The mitochondrial content of glutathione disulfide, as indicator of reactive oxygen formation determined 6 h after acetaminophen, increased from 2.8 +/- 0.6% in controls to 23.5 +/- 5.1%. A high dose of allopurinol (100 mg/kg) strongly attenuated acetaminophen protein-adduct formation and prevented the mitochondrial oxidant stress and liver injury after acetaminophen. Lower doses of allopurinol, which are equally effective in inhibiting xanthine oxidase, were not protective and had no effect on nitrotyrosine staining and acetaminophen protein adduct formation. In vitro experiments showed that allopurinol is not a direct scavenger of peroxynitrite. We conclude that there is vascular peroxynitrite formation during the first 2 h after acetaminophen treatment. On the other hand, reactive metabolites of acetaminophen bind to intracellular proteins and cause mitochondrial dysfunction and superoxide formation. Mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite formation and liver injury remains to be established.

AlphaMbeta2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to alphaM I domain that is divalent cation-sensitive.

The beta2 (CD18) leukocyte integrins play a key role in normal and inflammatory immune responses. In resting leukocytes, these receptors do not bind ligands. However, when leukocytes are exposed to an appropriate agonist, high-affinity ligand binding is achieved, presumably as a result of conformational changes in the integrin. In this study, we describe a novel monoclonal antibody, mAb 6C1, directed against the alphaM subunit, which directly induces adhesion of alphaMbeta2-transfected CHO cells to fibrinogen, ICAM-1, and iC3b. Induction of binding could also be accomplished by monovalent Fab fragments of mAb 6C1 at concentrations similar to that observed with intact IgG, demonstrating stimulation of adhesion was not because of receptor cross-linking at the cell surface. The binding of mAb 6C1 induces conformational changes in the receptor, as evidenced by the expression of an "activation reporter" epitope recognized by mAb 24. The binding of mAb 6C1 is modulated by divalent cations. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding. A unique distinction of mAb 6C1 is localization of its epitope to the alphaM I domain. The alphaM I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of alphaMbeta2 ligand-binding affinity. Thus, these studies have identified a novel alphaM I domain activation epitope of alphaMbeta2 and support the idea that the I domain modulates the activational state of the beta2 integrins.

Protection against TNF-induced liver parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice.

Excessive apoptotic cell death is implicated in a growing number of acute and chronic disease states. Caspases are critical for the intracellular signaling pathway leading to apoptosis. The aim of this investigation was to evaluate the efficacy and the mechanism of action of the novel caspase inhibitor CV1013 in a well-characterized model of TNF-induced apoptosis. Administration of 700 mg/kg galactosamine/100 microg/kg endotoxin (Gal/ET) induced hepatocellular apoptosis in C3Heb/FeJ mice as indicated by increased caspase-3 activity (706% above controls) and enhanced DNA fragmentation (3400% above controls) at 6 h. In addition, apoptosis was aggravated by the neutrophil-induced injury at 7 h (ALT activities: 4220 +/- 960 U/L and 48 +/- 4% necrosis). All animals died 8-12 h after Gal/ET treatment from shock and liver failure. A dose of 10 or 1 mg/kg of CV1013 administered three times (3, 4.5, and 5.5 h after Gal/ET) effectively prevented caspase-3 activation and parenchymal cell apoptosis at 6 h as well as the subsequent neutrophil-induced aggravation of the injury at 7 h after Gal/ET treatment. Animals treated with 10 mg/kg CV1013 survived for 24 h without liver injury. CV1013 reduced the processing of caspase-3 and caspase-8. This suggests that CV1013 may have inhibited the small amount of active caspase-8 generated at the receptor level. Because of the multiple amplification loops used to activate the entire caspase cascade, blocking the initial intracellular signal by CV1013 was highly effective in preventing apoptotic cell death. CV1013 has therapeutic potential for disease states with excessive apoptosis.

Protection against Fas receptor-mediated apoptosis in hepatocytes and nonparenchymal cells by a caspase-8 inhibitor in vivo: evidence for a postmitochondrial processing of caspase-8.

Lymphocytes can kill target cells including hepatocytes during various inflammatory diseases by Fas receptor-mediated apoptosis. Caspase-8 is activated at the receptor level, thereby initiating the processing of downstream effector caspases. The aim of this study was to investigate the time course of caspase-8 activation and to evaluate the efficacy of the caspase-8 inhibitor IETD-CHO in a model of Fas-induced apoptosis in vivo. C3Heb/FeJ mice were treated with the anti-Fas antibody Jo-2 (0.6 mg/kg). Western blot analysis demonstrated increased cytochrome c in the cytosol (20 min), which was followed by the progressive activation of caspase-3, -9 (40-120 min), and caspase-8 (120 min). At 90 and 120 min, extensive hemorrhage was observed, indicating damage to sinusoidal lining cells. In addition, high plasma ALT levels (997 +/- 316 U/L) and histological evaluation indicated severe parenchymal cell injury. Parenchymal and nonparenchymal cells showed a similar increase in caspase-3 activity and DNA fragmentation. Treatment with IETD-CHO (10 mg/kg) attenuated the increase in caspase-3 activity and DNA fragmentation by 80-90% and completely prevented hemorrhage and parenchymal cell damage. IETD-CHO also prevented the early release of mitochondrial cytochrome c and the processing of caspase-3, -8, and -9. Thus, our data support the hypothesis that Fas-mediated apoptosis is dependent on caspase-8 activation in hepatocytes and nonparenchymal cells. However, the bulk of procaspase-8 is processed late, suggesting that only a small amount of procaspase-8 may actually be activated at the Fas receptor. This initial signal may be amplified by further activation of caspase-8 by effector caspases, i.e., after mitochondrial activation. Caspase-8 is a promising therapeutic target for inhibition of Fas-mediated apoptosis.

The hepatic inflammatory response after acetaminophen overdose: role of neutrophils.

Acetaminophen overdose induces severe liver injury and hepatic failure. There is evidence that inflammatory cells may be involved in the pathophysiology. Thus, the aim of this investigation was to characterize the neutrophilic inflammatory response after treatment of C3Heb/FeJ mice with 300 mg/kg acetaminophen. A time course study showed that neutrophils accumulate in the liver parallel to or slightly after the development of liver injury. The number of neutrophils in the liver was substantial (209 +/- 64 PMN/50 high-power fields at 12 h) compared to baseline levels (7 +/- 1). Serum levels of TNF-alpha and the C-X-C chemokines KC and MIP-2 increased by 28-, 14-, and 295-fold, respectively, over levels found in controls during the injury process. In addition, mRNA expression of MIP-2 and KC were upregulated in livers of acetaminophen-treated animals as determined by ribonuclease protection assay. However, none of these mediators were generated in large enough quantities to account for neutrophil sequestration in the liver. There was no upregulation of Mac-1 (CD11b/ CD18) or shedding of L-selectin on circulating neutrophils. Moreover, an anti-CD18 antibody had no protective effect against acetaminophen overdose during the first 24 h. These results indicate that there is a local inflammatory response after acetaminophen overdose, including a substantial accumulation of neutrophils in the liver. Because of the critical importance of beta2 integrins for neutrophil cytotoxicity, these results suggest that neutrophils do not contribute to the initiation or progression of AAP-induced liver. The inflammation observed after acetaminophen overdose may be characteristic for a response sufficient to recruit neutrophils for the purpose of removing necrotic cells but is not severe enough to cause additional damage.

Expression of a structural domain of the beta 2 subunit essential for alpha M beta 2 ligand recognition.

The beta2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of beta2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the alpha and beta subunit. As an approach to identify and characterize regulatory domains of the beta2 subunit, we have generated two different subdomains of the beta2 subunit for expression on the surface of mammalian cells through a phosphatidyl-inositol glycan anchor. The first subdomain contains the putative beta2 MIDAS motif implicated in ligand binding [beta2(LB)], whereas the second beta2 subdomain contains the cysteine-rich region [beta2(CR)]. Cells expressing alphaM and beta2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant alphaMbeta2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete alphaMbeta2 heterodimer. In contrast, cells expressing alphaMbeta2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected alpha or beta chains alone failed to adhere to immobilized iC3b. These results indicate that along with alphaM, the beta2(LB) subdomain contains the sufficient components within the beta2 subunit essential for ligand recognition. These findings support the hypothesis that the beta2 subunit cooperates with site(s) within the alphaM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction.

Transcriptional activation of vascular cell adhesion molecule-1 gene in vivo and its role in the pathophysiology of neutrophil-induced liver injury in murine endotoxin shock.

Polymorphonuclear leukocytes (neutrophils) can cause hepatic parenchymal cell injury during endotoxin (ET) shock. Because adhesion molecules are critical for inflammatory cell damage, the role of vascular cell adhesion molecule-1 (VCAM-1) was studied in the pathophysiology of ET shock. ET-sensitive mice (C3Heb/FeJ) were treated with 700 mg/kg galactosamine in combination with 100 microg/kg Salmonella abortus equi ET, 15 microg/kg TNF-alpha, or 13 to 23 microg/kg IL-1. VCAM-1 mRNA formation was strongly activated in animals treated with ET, TNF-alpha, or IL-1. In contrast, only TNF-alpha and IL-1, not ET, induced VCAM-1 gene transcription in livers of ET-resistant mice (C3H/HeJ). Immunohistochemistry and isolation of liver cells during endotoxemia indicated that VCAM-1 mRNA and protein were only formed in endothelial cells and Kupffer cells, not in hepatocytes. Galactosamine/ET induced neutrophil accumulation in sinusoids (515 +/- 30 neutrophils/50 high power fields) followed by transmigration at 7 h. At that time, severe liver injury was observed (necrosis, 53 +/- 5%). An anti-VCAM-1 Ab (3 mg/kg) attenuated the area of necrosis by 60%. The Ab reduced neutrophil transmigration by 84%, but had no effect on the total number of cells in the liver vasculature. Flow cytometric analysis identified the presence of very late Ag-4 on mouse peripheral neutrophils. Our data demonstrated cytokine-dependent VCAM-1 gene transcription and protein expression in the liver during endotoxemia. Neutrophils were able to use very late Ag-4/VCAM-1 interactions to transmigrate into liver parenchyma in vivo. Preventing transmigration by blocking VCAM-1 protected hepatocytes against neutrophil-induced injury.

Identifying the putative metal ion-dependent adhesion site in the beta2 (CD18) subunit required for alphaLbeta2 and alphaMbeta2 ligand interactions.

We have previously demonstrated that Asp134 and Ser136 of the beta2 subunit are essential for alphaLbeta2 and alphaMbeta2 ligand recognition. It has been proposed that these residues may be part of a metal ion-dependent adhesion site (MIDAS) within the beta subunit homologous to the alphaM I domain MIDAS structure (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638). In the present study, we evaluated the role of additional candidate metal ion-coordinating residues in the beta2 subunit in ligand interactions. Cells bearing the recombinant alphaLbeta2 or alphaMbeta2 mutant(s) were tested for the ability to bind to immobilized ligands. Alanine substitution at Asp232 in beta2 produced a complete loss in the capacity of both alphaLbeta2 and alphaMbeta2 to support cell adhesion and suppressed the expression of a divalent cation-dependent conformation recognized by mAb 24. Alanine substitution at Glu235 differentially affected receptor function dependent upon the co-transfected alpha subunit. Cells expressing alphaLbeta2 with a substitution at Glu235 failed to adhere to intercellular adhesion molecule 1 (ICAM-1) but did retain the capacity to bind mAb 24. Moreover, cells expressing alphaMbeta2 with a substitution at Glu235 failed to adhere to fibrinogen or ICAM-1 and did not bind mAb 24. However, these cells did retain the capacity to adhere to iC3b following antibody-induced activation. These results implicate Asp232 and Glu235, along with Asp134 and Ser136, in ligand binding function of alphaLbeta2 and alphaMbeta2. These findings provide evidence in support of the existence of a MIDAS structure in beta2 analogous to that seen in the alphaM I domain.

Distinct ligand binding sites in the I domain of integrin alpha M beta 2 that differentially affect a divalent cation-dependent conformation.

The I domains of the leukocyte beta 2 integrins have been shown to be essential for ligand recognition. Amino acid substitutions of Asp140 and Ser142, which reside in a conserved cluster of oxygenated residues, abrogate divalent cation ligand binding function of alpha M beta 2. Presently, we evaluated the role of two I domain regions in alpha M beta 2 ligand recognition: 1) the conserved cluster of oxygenated residues (Asp134, Asp140, Ser142, and Ser144) and 2) a 7-amino acid region (Phe246-Tyr252), conserved in alpha M and alpha X but absent in alpha L of the beta 2 integrins. Recombinant alpha M beta 2 was expressed on COS-7 cells, and function was assessed by iC3b recognition. Alanine substitution at position Asp140, Asp140/Ser142, Ser142, or Ser144 produced a complete loss in the capacity of alpha M beta 2 to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp248 or Tyr252 or deletion of Phe246-Tyr252 abolished iC3b ligand recognition as well as the binding of a blocking antibody. In contrast, these mutations did not affect the binding of the cation-dependent epitope. These data implicate a second region within the I domain important for alpha M beta 2 ligand binding function and suggest that this region does not affect a divalent cation-dependent conformation of alpha M beta 2.

Beta 2 (CD18) mutations abolish ligand recognition by I domain integrins LFA-1 (alpha L beta 2, CD11a/CD18) and MAC-1 (alpha M beta 2, CD11b/CD18).

The "I" domains of the beta 2 (CD18) leukocyte integrins are implicated in ligand binding function. Moreover, rather than recognizing linear peptide sequences, this class of integrins generally recognizes multiple discontinuous sites on immunoglobulin superfamily adhesion receptors. A conserved cluster of oxygenated residues is involved in ligand recognition by beta 1 and beta 3 integrins. In the present study, we evaluated the role of this region in the I domain-containing beta 2 integrins. Recombinant alpha L beta 2 (LFA-1, CD11a/CD18) and alpha M beta 2 (MAC-1, CD11b/CD18) were expressed on COS cells, and function was assessed by adhesion to ICAM-1 or iC3b, respectively. Alanine substitution at position Asp134 or Ser136 in beta 2 produced a complete loss in the capacity of both alpha L beta 2 and alpha M beta 2 to support cell adhesion. In contrast, substitution at Asp128 or Ser138 resulted in loss of beta 2 surface expression when co-transfected with alpha L (CD11a) or alpha M (CD11b). These data provide the first evidence for involvement of the beta 2 subunit in ligand binding to I domain integrins.

Mutation of a ligand binding domain of beta 3 integrin. Integral role of oxygenated residues in alpha IIb beta 3 (GPIIb-IIIa) receptor function.

A single amino acid substitution in beta 3 (Asp119 --> Tyr) abrogates the ligand binding function of beta 3 integrins and alters the divalent cation conformation of the platelet integrin alpha IIb beta 3 (GPIIb-IIIa). This aspartic acid residue resides within a conserved cluster of oxygenated residues that may provide ligands for the coordination of divalent cations. To assign function to the other oxygenated residues in this group (Ser121, Ser123, Asp126, Asp127, and Ser130), each of these amino acids in beta 3 was individually substituted by alanine. None of these amino acid substitutions altered heterodimer formation or surface expression. However, the substitutions had differential effects on receptor function. Substitution at positions Asp119 or Ser121 produced a complete loss of receptor function. Cells expressing these mutants failed to adhere to fibrinogen, failed to bind activation-independent ligand-mimetic peptides, and did not bind the ligand-mimetic mAb PAC1 following activation of the receptor. Similarly, cells expressing beta 3 with a substitution at Ser123 also failed to adhere to fibrinogen and did not bind RGD peptide or mAb PAC1. These cells did retain the capacity to bind an alpha IIb beta 3-specific, high affinity peptidomimetic, but occupancy did not induce the conformational change from resting to activated state observed following occupancy of the wild type receptor. Substitution at positions Asp126, Asp127, or Ser130 had no effect on ligand binding function. These data indicate that Asp119, along with Ser121 and Ser123, plays an integral role in the ligand binding function of alpha IIb beta 3.

Characterization of a gain of function mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa).

Integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) is a prototype of integrins involved in cellular adhesive functions. As part of a structure-function analysis of this molecule, we constructed a mutant, designated alpha IIb beta 3 (beta 1-2), by replacing 6 amino acids within a putative ligand binding domain of the beta 3 subunit with sequences derived from beta 1. The alteration did not affect the capacity of beta 3(beta 1-2) to combine with transfected alpha IIb, nor did it cause it to combine with endogenous alpha 5. Integrin alpha IIb beta 3(beta 1-2) was in a "resting" state on Chinese hamster ovary cells as judged by minimal binding of an activation-specific anti-alpha IIb beta 3, PAC1. Nevertheless, cells expressing alpha IIb beta 3(beta 1-2) spontaneously bound fibrinogen with low affinity (Ka = (4.85 +/- 0.84) x 10(6) M-1). Activation with an anti-beta 3 antibody (monoclonal antibody 62) resulted in a 10-fold increase in fibrinogen binding affinity (Ka = (4.55 +/- 0.77) x 10(7) M-1), which was 3-fold greater than fibrinogen binding to activated wild type alpha IIb beta 3 (Ka = (1.66 +/- 0.33) x 10(7) M-1, F = 7.46, p = 0.008). The mutant receptor also bound fibrinogen mimetic peptide ligands with enhanced affinity as measured by the conformation-specific antibody, anti-LIBS1. This indicates that the increased affinity for fibrinogen was caused by enhanced interaction of alpha IIb beta 3(beta 1-2) with known recognition sequences in fibrinogen. Thus, this gain of function mutant augments ligand binding function, supporting a role for this region of the beta subunit in ligand binding to integrins.

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.

Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIB-IIIA) dependent homotypic and heterotypic cell-cell interactions.

The aggregation of cells bearing recombinant integrin alpha IIb beta 3 (platelet GPIIb-IIIa) has been analyzed by two-color flow cytometry. As in normal platelets, aggregation requires functional alpha IIb beta 3, "activation" of alpha IIb beta 3, and fibrinogen (fg) binding to alpha IIb beta 3. Cellular aggregation required that both interacting cells express functional alpha IIb beta 3, because a binding defective mutant, alpha IIb beta 3 (D119----Y), failed to support interaction with wild type alpha IIb beta 3-bearing cells. In addition, cells bearing resting alpha IIb beta 3 were incorporated into aggregates formed by cells bearing a constitutively active mutant, alpha IIb beta 3 (beta 1-2), indicating that only one of the cells in an interacting pair must be activated. Finally, heterotypic interactions occurred between cells bearing activated alpha IIb beta 3 and cells bearing alpha V beta 3, a fg-binding integrin present on endothelial and tumor cells. Thus, ligand bridging between fg-binding integrins represents a mechanism of cell-cell interaction, cells bearing resting alpha IIb beta 3 (e.g., resting platelets) may be incorporated into aggregates formed by cells bearing activated alpha IIb beta 3, and alpha IIb beta 3 mediates heterotypic interactions with cells bearing other fg receptors.

The specificity of 130-kDa leech sensory afferent proteins is encoded by their carbohydrate epitopes.

From early development through adulthood in the leech, sensory afferents, glial cells, and connective tissue express different epitopes located on a group of 130-kDa glycoproteins. The sensory epitope [reactive with monoclonal antibody (mAb) Lan3-2] is shared by the peripheral sensory afferents of different sensory modalities. In contrast, three other immunocytochemically distinct epitopes (reactive with mAbs Laz2-369, Laz7-79, and Laz6-212) differentiate these sensory afferents according to their sensory modalities. The glial epitope (mAb Laz6-297) is expressed on all macroglial processes, and the connective tissue epitope (mAb Laz9-84) is located on connective tissue surrounding the CNS, as well as in the peripheral tissues. The hydrophilic-hydrophobic nature of the 130-kDa sensory afferent and glial proteins was determined by phase separation with Triton X-114 and hypoosmotic extraction. They behave as peripheral membrane proteins. Deglycosylation of 130-kDa glycoproteins with N-Glycanase or preincubation of their respective mAbs with alpha-methylmannoside showed that the sensory epitope contains mannose, whereas the modality epitopes are of an undefined carbohydrate character. Immunoprecipitation and a peptide mapping experiment confirmed the existence of four distinct sensory afferent epitopes. Previous studies provided evidence that the mannose-containing Lan3-2 epitope mediates normal sensory afferent growth in the synaptic neuropile. We, therefore, postulate that the carbohydrate epitopes on sensory afferent glycoproteins participate in synapse formation.

Carbohydrate epitopes involved in neural cell recognition are conserved between vertebrates and leech.

We are reporting on the evolutionary conservation of carbohydrate epitope families from vertebrate to leech. 1) The sulfated L2/HNK-1 carbohydrate epitope (Abo T, Balch CM (1981): J Immunol 127:1024-1029; Kruse J, Mailhammer R, Wernecke H, Faissner A, Timpl R, Schachner M (1984): Nature 311:153-155) is detected on glycoproteins of leech neurons using monoclonal antibodies (mAbs) L2 (336) and HNK-1. 2) Three rat mAbs, L3, L4, and L5, bind to leech nerve and muscle. The L3, L4, and L5 epitopes are localized to a group of mannosidic leech glycoproteins originally identified through mAbs Lan3-2 (Hogg N, Flaster M, Zipser B (1983): J Neurosci Res 9:445-457 and Laz6-189 (McRorie JW III, Zipser B (1988): "Cell Culture Approaches to Invertebrate Neuroscience." London: Academie Press, pp 33-52. MAb Lan3-2, which binds to a mannosidic epitope of the 130 kD sensory protein, has recently been shown to perturb the penetration of sensory afferents into the synaptic area of the central neuropile (Zipser B, Morell R, Bajt ML (1989): Neuron 3:621-630). The L3, L4, and L5 mAbs have been described to recognize different mannosidic epitopes on glycoproteins, some of which have been identified as neural cell adhesion molecules, and on astrocyte-specific proteoglycan from mouse brain (Kücherer A, Faissner A, Schachner M (1987): J Cell Biol 104:1597-1602; Fahrig T, Schmitz B, Weber D, Kücherer-Ehret A, Faissner A, Schachner M (1990): Eur J Neurosci 2:153-161; Streit A, Faissner A, Gehrig B, Schachner M (1990): J Neurochem In Press). The superposition of five different mannosidic epitopes on the axons of sensory afferents suggests complex, concerted participation of mannosidic epitopes in neuronal pathfinding and target recognition.

Defasciculation as a neuronal pathfinding strategy: involvement of a specific glycoprotein.

Leech sensory afferents change their growth behavior as they enter the CNS. Arriving from the periphery in fasciculated tracts, they abruptly defasciculate and expand into diffuse trees before reassembling into four distinct central tracts. In the organ-cultured germinal plate, growing sensory afferents were incubated with monovalent Fab fragments of the Lan3-2 antibody, which recognizes a 130 kd sensory neuron protein by its mannose epitope. Very low concentrations of Lan3-2 (6 and 12 nM) specifically inhibited the central defasciculation of sensory afferents, which then continued growing as a single tract. In contrast, monoclonal antibody Lan3-6, which binds to an internal sensory antigen, failed to yield the same effect. These observations suggest that this sensory neuron 130 kd surface glycoprotein participates in a developmentally significant heterophilic interaction specific for the CNS.

An analysis of factors responsible for resorption of embryos in cisplatin-treated rats.

Pregnant rats were injected ip with 4 or 7 mg cisplatin/kg on gestation day (gd) 6 to study its effect on embryonal resorption. Serum concentrations of prolactin, luteinizing hormone (LH), and progesterone were determined by radioimmunoassay in pregnant rats, and related to the effects of cisplatin on the maintenance of pregnancy. The nocturnal prolactin surge on gd 9 was abolished in cisplatin-treated rats. Within 3 days after drug injection, LH concentrations decreased 39%, while serum progesterone decreased 63% by Day 10. A histochemical study of 20 alpha-hydroxysteroid dehydrogenase activity revealed no enzyme activity by gd 10. It is proposed that the cause of cisplatin-related embryonal resorption in rats may be due to decreases in hormone concentrations observed after drug treatment.