Activation-induced mobilization of secretory vesicles in bovine neutrophils.

OBJECTIVE: To characterize mobilization of secretory granules in bovine neutrophils. SAMPLE POPULATION: Neutrophils obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: Mobilization of secretory granules in bovine neutrophils was determined by measuring changes in cell-surface alkaline phosphatase activity on cells treated with various inflammatory mediators. Subcellular distribution of the alkaline phosphatase activity was determined by analysis of bovine neutrophil homogenates fractionated on density gradients. RESULTS: Alkaline phosphatase-containing secretory granules of bovine neutrophils were readily mobilized by a number of inflammatory agents, including platelet-activating factor, interleukin-8, tumor necrosis factor-alpha, lipopolysaccharide, leukotriene B4, and zymosan-activated plasma. In contrast, N-formyl-methionyl-leucyl-phenylalanine did not have a significant effect. Phorbol myristate acetate induced a biphasic response with up-regulation of cell-surface alkaline phosphatase at low doses and a return to baseline or even a reduction in cell-surface alkaline phosphatase at higher doses (> or = 10 ng/ml). Subcellular fractionation of bovine neutrophil homogenates revealed that alkaline phosphatase activity resided in light-density membrane vesicles (ie, location of secretory granules), which were distinct from specific, azurophil, and large granules. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine neutrophils respond to various inflammatory mediators by mobilizing alkaline phosphatase-containing secretory granules. This suggests that the process is an important early step in the host-defense response of bovine neutrophils.

A carbohydrate neoepitope that is up-regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation.

The expression of cell-surface antigens can delineate specific leucocyte developmental or functional stages. For example, certain membrane glycoproteins are expressed selectively on leucocyte subsets only after activation. Leucocyte activation can also induce changes in carbohydrate epitopes expressed on surface antigens. In the present studies, we report on a novel monoclonal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohydrate epitope expressed on human leucocyte membrane proteins. Characterization of mAb 13.22 specificity by immunoblotting showed that it recognized proteins of MW approximately 95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N-glycosidase F but not by endoglycosidase H or fucosidase, demonstrating that it is an N-linked carbohydrate antigen. Interestingly, immunoblot staining was enhanced after neuraminidase treatment, suggesting that the antibody epitope might also be partially masked by sialic acid. In resting leucocytes, the mAb 13.22 antigen was expressed strongly on neutrophils, while dull staining was present on monocytes, and no lymphocyte staining was observed. In marked contrast, treatment of leucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cells) as well as up-regulated staining more than 18-fold on monocytes. Activation of lymphocytes in culture with phytohaemagglutinin or concanavalin A also unmasked the mAb 13.22 neoepitope on approximately 37% of the CD45RO+ lymphocytes. Furthermore, analysis of leucocytes collected from the synovial fluid of patients with rheumatoid arthritis showed that approximately 18% of the lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, our results suggest that the mAb 13.22 carbohydrate neoepitope could represent a physiologically relevant marker that is up-regulated on leucocyte subsets during the inflammatory response.

Host defense function in neutrophils from the American bison (Bison bison).

Selected host defense functions of neutrophils isolated from American bison (Bison bison) were characterized and compared with those of cattle (Bos taurus). Bison neutrophils had a robust chemotactic response to both IL-8 and LTB(4), with maximal responses occurring at 10(-7) M (IL-8) and 10(-8) M (LTB(4)). The magnitude of the chemotactic response to IL-8 was similar in bison and bovine neutrophils (except at 10(-7) M IL-8, where bison had a stronger response). In response to LTB(4), bison neutrophils had a much stronger chemotaxis at both 10(-8) and 10(-7) M than did bovine cells. Production of reactive oxygen species (ROS) in response to phorbol myristate acetate (PMA) and opsonized zymosan (OpZ) was similar between bison and bovine neutrophils. However, the production of ROS in bison neutrophils stimulated with OpZ was primarily intracellular, while extracellular release of ROS was evident in bovine neutrophils stimulated with OpZ. Like bovine neutrophils, bison neutrophils did not generate a respiratory burst in response to fMLF. Granules prepared from bison neutrophils had potent direct killing action on the Gram-negative bacteria Escherichia coli but failed to kill the Gram-positive bacteria Staphylococcus aureus and, at intermediate doses, actually had a permissive effect for this bacteria. Thus, bison neutrophils have potent host defense capabilities similar in quality to those of bovine neutrophils; however, unique differences are present, which may allow bison neutrophils to respond to the distinct immunological challenges that bison encounter.

Cloning and characterization of bovine low molecular weight GTPases (Rac1 and Rac2) and rho GDP-dissociation inhibitor 2 (D4-GDI).

GTPases of the Rho family play important roles in human leukocyte signal transduction pathways; however, little is known about the function of these proteins in bovine cells. In the present studies, we isolated molecular clones of bovine Rac1, Rac2, and the Rac/Rho GTPase regulatory protein D4-GDP dissociation inhibitor (D4-GDI) from a bovine bone marrow cDNA library. These clones contained complete open reading frames, encoding 192, 192, and 200 amino acids, respectively. Comparison of the bovine amino acid sequences with those of other species demonstrated a high degree of identity of these proteins across all species, suggesting that these proteins likely play conserved functional roles in bovine leukocyte signal transduction pathways. Comparative Western blotting of these proteins in human and bovine neutrophil cytosol demonstrated that Rac2 was the predominant Rac species and that D4-GDI was the predominant GDI species in bovine neutrophil cytosol. Despite the high degree of homology between human and bovine Rac2, some of the anti-peptide antibody probes prepared against human Rac2 failed to recognize the bovine homologue. We also showed by subcellular fractionation techniques that Rac2 is localized primarily to the cytosolic compartment of resting bovine neutrophils, but is translocated to the plasma membrane after stimulation with PMA. These findings suggest that Rac2 does play a role in bovine neutrophil activation. In addition, these data will be helpful in developing more specific probes for investigating the role of these proteins in bovine leukocyte signal transduction pathways and for studying various inflammatory diseases in cattle.

Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs.

Neutrophils play an essential role in bovine cellular host defense, and compromised leukocyte function has been linked to the development of respiratory and mucosal infections. During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. Two of the essential cytosolic components of the NADPH oxidase are p47-phox and p67-phox. Currently, only the human and murine homologs of these proteins have been sequenced. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and express bovine p47-phox and p67-phox. Using polymerase chain reaction (PCR) cloning techniques and a bovine bone marrow cDNA library, we have cloned both of these bovine NADPH oxidase cytosolic components. Comparison of the bovine sequences with those of the human and murine homologs showed that they were highly conserved, but also revealed important information regarding key structural features of p47-phox and p67-phox, including location of putative phosphorylation sites. Functional expression of bovine p47-phox and p67-phox showed that these proteins could substitute for the human proteins in reconstituting NADPH oxidase activity in a cell-free assay system, again demonstrating the high degree of conservation between human and bovine homologs. This study greatly contributes to our understanding of the potential structural/functional regions of p47-phox and p67-phox as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases.

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

Antibody imprint of a membrane protein surface. Phagocyte flavocytochrome b.

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody "imprint" of the Cyt b surface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phage-display analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29TAGRF33 and 183PQVNPI188 from discontinuous regions of p22. Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cyt b surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.

Extensive contact between Gi2 and N-formyl peptide receptor of human neutrophils: mapping of binding sites using receptor-mimetic peptides.

The N-formyl peptide receptor (FPR) of human neutrophils is a member of the G protein-coupled receptor (GPCR) superfamily. Sites on agonist-occupied FPR involved in binding the Gi2 protein were investigated by competition with synthetic receptor-mimetic peptides. Twenty-three synthetic FPR-mimetic and control peptides were tested for their ability to disrupt functionally active complexes of FPR and Gi2 in octyl glucoside, assayed by changes in sedimentation rates of FPR in detergent-containing sucrose gradients. GPCRs are thought to contain seven transmembrane segments with three cytoplasmic connecting loops and a cytoplasmic tail. Only certain peptides from regions in or adjoining each of the four predicted cytoplasmic domains of the 350 amino acid FPR, including the first cytoplasmic loop, were able to disrupt the reconstituted FPR-Gi2 complex. The IC50s of the peptides that were able to fully disrupt the FPR-Gi2 complex ranged from 20 microM (C2W 122-134) to 1.4 mM (C3A 230-246), a range similar to peptide inhibition of other G protein-coupled receptor-G protein interactions. Detergent concentrations above and below the critical micelle concentration had no effect on the activity of even the most hydrophobic peptide, C3B, and there was no apparent correlation of activity with hydrophobic moment, hydrophilic index, or net charge of the peptides. Control peptides from irrelevant proteins with similar physical properties and FPR extracellular domains did not dissociate the reconstituted FPR-Gi2 complex up to 5 mM, the highest concentration tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of complex formation between Gi2 and octyl glucoside solubilized neutrophil N-formyl peptide chemoattractant receptor by sedimentation velocity.

The reversible formation of complexes between N-formyl peptide chemoattractant receptor (FPR) and Gi2 protein was analyzed by velocity sedimentation in linear sucrose density gradients. FPR complexed with heterotrimeric Gi2, sediments at different rate than uncomplexed FPR and the two forms have apparent sedimentation coefficients of 7S and 4S, respectively. The biochemical variables important for the reconstitution of the 7S complex from the 4S receptor and Gi2 were studied. The formation of 7S was saturable with Gi2 and addition of excess Gi did not cause oligomerization. The reconstituted 7S complex was stable under a variety of conditions including octyl glucoside concentrations below and above the critical micellar concentration. The optimum pH for the reconstitution is between 7 and 9, where the 4S and 7S species sedimented reproducibly, at distinct positions in the gradient. Below pH 6 both the 4S and the 7S species appear to undergo denaturation and form precipitates. Magnesium ions have no significant effect on the sedimentation of either forms of FPR. Reconstitution was stable up to a NaCl concentration of 0.2 M. At 1 M NaCl reconstitution was inhibited and at 3 M salt FPR aggregated. Since guanine nucleotides GTP, GTP gamma S, GDP beta S selectively dissociated the 7S complex in a concentration-dependent manner and adenine nucleotides had no effect, we conclude that the FPR-Gi2 system displays a vacant guanyl nucleotide binding site, the hallmark of a functional guanine nucleotide exchange complex. Moreover, our results indicate that the reconstitution of FPR-Gi2 complexes is reproducible at physiologically relevant conditions, shows selectivity, specificity, and biochemically functional properties consistent with a specific and functional interaction between solubilized FPR and G protein.

Functional molecular complexes of human N-formyl chemoattractant receptors and actin.

When human neutrophils become desensitized to formyl peptide chemoattractants, the receptors (FPR) for these peptides are converted to a high affinity, GTP-insensitive form that is associated with the Triton X-100-insoluble membrane skeleton from surface membrane domains. These domains are actin and fodrin-rich, but G protein-depleted suggesting that FPR shuttling between G protein-enriched and depleted domains may control signal transduction. To determine the molecular basis for FPR interaction with the membrane skeleton, neutrophil subcellular fractions were screened for molecules that could bind photoaffinity-radioiodinated FPR solubilized in Triton X-100. These receptors showed a propensity to bind to a 41- to 43-kDa protein band on nitrocellulose overlays of SDS-PAGE-separated cytosol and plasma membrane fractions of neutrophils. This binding, as well as FPR binding to purified neutrophil actin, was inhibited 50% by 0.6 microM free neutrophil cytosolic actin. Addition of greater than 1 microM G-actin to crude or lectin-purified Triton X-100 extracts of FPR from neutrophil membranes increased the sedimentation rate of a significant fraction of FPR two to three fold as measured by velocity sedimentation in Triton X-100-containing linear sucrose density gradients. Addition of anti-actin antibodies to FPR extracts caused a concentration-dependent immunoprecipitation of at least 65% of the FPR. More than 40% of the immunoprecipitated FPR was specifically retained on protein A affinity matrices. Membrane actin was stabilized to alkaline washing when membranes were photoaffinity labeled. Conversely, when purified neutrophil cytosolic actin was added to membranes or their digitonin extracts, after prior depletion of actin by an alkaline membrane wash, photoaffinity labeling of FPR was increased two- to fourfold with an EC50 of approximately 0.1 microM actin. We conclude that FPR from human neutrophils may interact with actin in membranes to form Triton X-100-stable physical complexes. These complexes can accept additional G-actin monomers to form higher order molecular complexes. Formation of FPR-actin complexes in the neutrophil may play a role in the regulation of chemoattractant-induced activation or actin polymerization.

Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

Standard setting in optometric clinical skills examinations.

Objective-based clinical skills examinations have become an accepted method of assessing minimal competency for licensure in optometry. This paper reviews key elements of Clinical Skills Examination (CSE), and how standard setting for CSEs can be improved.