Most strains ofgroup B streptococci (GBS) elaborate a cell surface-associated enzyme that rapidly inactivates the human complement-derived chemoattractants C5a and C5ad,g by cleaving the His-Lys bond at positions 67 and 68 in the C5a molecule. We have suggested that rapid inactivation of C5a and C5ader,g by this enzyme, called C5a-ase, can hinder the inflammatory response at sites of GBS infection. We tested the ability of GBS C5a-ase to inactivate C5a preparations from various animal species to determine the proper species for studying the role of GBS C5a-ase in the pathogenesis of GBS infections. Exposure of C5a preparations from humans, monkeys, and cows to GBS caused inhibition of C5a functional activity as measured by the ability of C5a to stimulate human polymorphonuclear leukocyte (PMN) adherence and human PMN chemotaxis. Bovine PMN chemotaxis to bovine C5a was also abolished after exposure of bovine C5a to GBS. In contrast, mouse, rat, guinea pig, rabbit, pig, and sheep C5a preparations retained full functional activity after exposure to GBS as measured by chemotaxis of human PMNs, PMNs from the same animal species, or both. These data suggest that there are structural differences between C5a proteins from different species which alter their susceptibility to GBS C5a-ase and indicate that most commonly used animal models of human GBS infection are inadequate for detection of a contribution of GBS C5a-ase to GBS virulence.

We investigated the mechanism of beta 1 integrin-mediated adherence of stimulated granulocytic HL60 cells to fibronectin using a monoclonal antibody (15/7) that recognizes beta 1 integrins only when the receptors are active for ligand binding. Phorbol myristate acetate (PMA) stimulated expression of the 15/7 epitope on granulocytic HL60 by nearly fivefold but had an insignificant effect on the expression of the epitope on undifferentiated HL60 cells. These results paralleled the effect of PMA on HL60 and granulocytic HL60 adhesion to fibronectin, indicating that activation of beta 1 integrins is important for beta 1-mediated adherence of granulocytic HL60 cells to fibronectin. Agonists that stimulate alpha 5 beta 1-dependent human polymorphonuclear leukocyte (PMN) adhesion to fibronectin (C5a and PMA) also upregulated the 15/7 epitope on purified human PMNs. Although PMA rapidly induces increased levels of filamentous actin (F-actin) in granulocytic HL60 cells and a decrease in F-actin levels in undifferentiated HL60 cells, depolymerization of the actin cytoskeleton with cytochalasin B did not affect increased expression of the 15/7 epitope on granulocytic HL60 cells. Cytochalasin B did, however, inhibit granulocytic HL60 adherence to fibronectin by 50%, demonstrating that actin polymerization is important for optimal beta 1-dependent granulocytic adherence.

We have compared the size, the binding to Concanavalin A (Con A), and the affinity for monoclonal antibody 1116NS-199 (Mab 19-9) among CA 19-9 molecules from sera of cystic fibrosis (CF) and pancreatic carcinoma patients and from sputum extracts. CA 19-9 molecules of two different sizes were found in all types of specimens by Sepharose 4B chromatography. While the smaller CA 19-9 molecule was predominant in CF patient sera, the larger molecule was associated with most of the sera from patients with pancreatic carcinoma. The majority of the sputum extracts contained the larger CA 19-9 molecule. All CA 19-9 molecules studied by Con A chromatography did not appear to bind to Con A, and almost 100% were found in the nonreactive fraction. The CA 19-9 molecules from sera of either CF or pancreatic carcinoma patients exhibited variable affinities for Mab 19-9, some approaching that of the standard curve but many also having lower affinities. The lowest affinity was displayed by CA 19-9 molecules from the sputum extract. It appears that development of more specific assays for CF and for carcinoma is possible if the correct CA 19-9 molecule is selected for antibody preparation and for use as standards.

Phorbol esters upregulate the functional affinity of beta 1 integrin receptors for fibronectin on human neutrophils and other leukocytes. We investigated the ability of phorbol myristate acetate (PMA) to stimulate the human promyelocytic cell line HL-60 to adhere to fibronectin, either in its undifferentiated state (HL60) or after dimethylsulfoxide-induced differentiation along the granulocytic pathway (dHL60). PMA stimulated little adherence of undifferentiated HL60 to fibronectin or to the 120-kD chymotryptic cell-binding domain (CBD) of fibronectin. In contrast, PMA stimulated dHL60 cells to rapidly adhere to both fibronectin- and to CBD-coated plastic. PMA- stimulated dHL60 adherence to fibronectin was largely mediated by both alpha 4 beta 1 and alpha 5 beta 1, whereas PMA-stimulated dHL60 adherence to CBD was largely mediated by alpha 5 beta 1. There was little contribution from beta 2 integrins to PMA-stimulated dHL60 adherence to fibronectin or CBD. The inability of undifferentiated HL60 to adhere to fibronectin and CBD did not result from lack of expression of alpha 4 beta 1 or alpha 5 beta 1 because HL60 and dHL60 express similar amounts of both alpha 4 beta 1 and alpha 5 beta 1 on their surface. In addition, 1 mmol/L Mn2+ induced similar amounts of alpha 5 beta 1-dependent adherence of both HL60 and dHL60, showing that alpha 5 beta 1 on undifferentiated HL60 is capable of binding to its ligand. These data suggest that activation of protein kinase C cannot functionally upregulate these beta 1 integrins on undifferentiated HL60 cells. The development of PMA-stimulated beta 1-dependent adherence after granulocytic differentiation of HL60 cells suggests that the differentiated HL60 cell is a useful model for investigating functional coupling of protein kinase C to beta 1 integrin in myeloid cells.