Michael-type reaction of ethyl bromodifluoroacetate with alpha,beta- unsaturated carbonyl compounds in the presence of copper powder.

We have reported that the reaction of ethyl bromodifluoroacetate (1) with alkenyl iodides in the presence of copper powder gives ethyl alkenyldifluoroacetates. As an extension of this reaction, reaction of 1 with Michael acceptors in the presence of copper powder was examined and found to give 1,4-addition products selectively, unless the acceptor has a group stabilizing a radical intermediate, such as a phenyl group.

Troglitazone suppresses cell growth of myeloid leukemia cell lines by induction of p21WAF1/CIP1 cyclin-dependent kinase inhibitor.

In a human eosinophilic leukemia cell line, EoL-1, cell proliferation was suppressed by 2-day treatment with troglitazone. EoL-1 cells treated with troglitazone were arrested and maintained in the G0/G1 phase in the cell cycle. This suppression correlated with the up-regulation of mRNA for p21WAF1/CIP1 cyclin-dependent kinase (Cdk) inhibitor. The inhibitory effects of troglitazone on cell proliferation and expression of p21 mRNA were observed in a human myelomonocytic cell line, U937, and a human myelomonoblastic cell line, KPB-M15. In addition, in EoL-1 cells, p21 protein was induced by troglitazone treatment and the induction was inhibited by protein synthesis inhibitor, cycloheximide. These data suggest that troglitazone inhibits cell proliferation in myeloid leukemia cell lines at least in part by induction of p21 Cdk inhibitor.

Stimulatory and inhibitory actions of lysophosphatidylcholine, depending on its fatty acid residue, on the phospholipase C/Ca2+ system in HL-60 leukaemia cells.

We examined the mechanism of action of lysophosphatidylcholine (LPC), which is suggested to be involved in the pathogenesis of atherosclerosis and inflammatory disorders, in HL-60 leukaemia cells. Extracellular 1-palmitoyl LPC increased the intracellular Ca2+ concentration in association with production of inositol phosphate. These actions of LPC were markedly inhibited by treatment of the cells with pertussis toxin and U73122, a phospholipase C inhibitor. The lipid-induced stimulation of the phospholipase C/Ca2+ system was also attenuated in the dibutyryl cAMP-induced differentiated (neutrophil-like) cells, in which phospholipase C activation induced by NaF or formyl-Met-Leu-Phe was enhanced. In contrast with the stimulatory action of 1-palmitoyl LPC, 1-stearoyl LPC was inhibitory for the phospholipase C/Ca2+ system stimulated by NaF as well as by 1-palmitoyl LPC or other Ca2+-mobilizing agonists. In a cell-free system, only an inhibitory effect on phospholipase C activity was observed even by 1-palmitoyl LPC; 1-stearoyl LPC was more inhibitive than 1-palmitoyl LPC. Taken together, these results suggest that atherogenic and inflammatory LPC exerts both stimulatory and inhibitory actions on the phospholipase C/Ca2+ system depending on the species of fatty acid residue of the lipid; the stimulatory effect is possibly mediated through G-protein-coupled receptors; the inhibitory effect might be caused by dysfunction of the components involved in the enzyme system owing to the amphiphilic nature of the lipid. 1-Palmitoyl LPC prefers the former receptor stimulation at least in intact cells, but 1-stearoyl LPC preferentially exerts the latter inhibitory action.

Downregulation of mRNA expression of Edg-3, a putative sphingosine 1-phosphate receptor coupled to Ca2+ signaling, during differentiation of HL-60 leukemia cells.

We measured the mRNA expression of the recently identified putative sphingosine 1-phosphate (S1P) receptors, i.e., Edg-1, AGR16/H218, and Edg-3, in HL-60 leukemia cells. Of these putative receptors, Edg-3 mRNA was abundantly expressed in undifferentiated HL-60 cells. Further, its mRNA expression was markedly downregulated by inducers of cell differentiation such as dibutyryl cAMP, retinoic acid, and 1alpha, 25-dihydroxyvitamin D3. The reduction of mRNA expression was associated with the attenuation of an S1P-induced increase in cytoplasmic free Ca2+ concentration. Thus, Edg-3, whose mRNA expression is downregulated during cell differentiation, may be responsible for the S1P-induced Ca2+ response in HL-60 leukemia cells.

Biosynthesis of B2-integrin, intracellular calcium signalling and functional responses of normal and CD18-deficient bovine neutrophils.

1Biosynthesis of CD11/CD18 in bovine leucocytes, intracellular Ca2+ ([Ca2+]i) signalling, chemiluminescent responses and membrane fluidity of neutrophils and the effects of D-mannose on neutrophils from control heifers and a heifer with bovine leucocyte adhesion deficiency (BLAD) were measured. The synthesis of CD11/CD18 complex was clearly detected in leucocytes from a normal heifer, but not in a BLAD-affected heifer. The transient phase of increased [Ca2+]i was clearly detected in neutrophils from a heifer with BLAD stimulated with opsonised zymosan, aggregated bovine immunoglobulin G or concanavalin A, whereas the sustained phase was deficient or significantly decreased compared with control heifers. [Ca2+]i signalling of neutrophils from control heifers and a heifer with BLAD stimulated with phorbol myristate acetate via an 11b/CD18-independent pathway showed no transient phase, and the subsequent increase in [Ca2+]i was almost identical in neutrophils from affected and control heifers. [Ca2+]i concentration and chemiluminescent responses of neutrophils from a control heifer were clearly decreased by treatment with anti-CD18 and anti-IgG antibodies. No differences in membrane fluidity were detected between neutrophils derived from control and CD18-deficient cattle. D-mannose binds mainly to Fc rather than CD18 receptors, and decreased Agg-IgG induced [Ca2+]i and the chemiluminescent response of neutrophils. The [Ca2+]i responses and Agg-IgG induced chemiluminescent responses of neutrophils from control heifers and a BLAD-affected heifer were inhibited by D-mannose. The characteristic changes of [Ca2+]i signalling and functional responses of B2-integrin-deficient neutrophils were demonstrated.

Involvement of pertussis toxin-sensitive GTP-binding proteins in sphingosine 1-phosphate-induced activation of phospholipase C-Ca2+ system in HL60 leukemia cells.

Exogenous sphingosine 1-phosphate (S1P) induced Ca2+ mobilization, in association with an increase in inositol polyphosphate production reflecting activation of phospholipase C in HL60 leukemia cells. The increase in intracellular Ca2+ concentration ([Ca2+]i) induced by S1P was inhibited by an appropriate treatment of the cells with pertussis toxin (PTX), U73122 (a phospholipase C inhibitor) or phorbol 12-myristate 13-acetate (PMA). In parallel with the Ca2+ response, these agents also inhibited inositol polyphosphate production. The S1P-induced Ca2+ response was also attenuated in the dibutyryl cAMP-induced differentiated cells, where GTP-binding protein-induced Ca2+ response suggested to be enhanced. Lysophosphatidic acid (LPA) also increased [Ca2+]i in the cels, but the maximal response was about half of that of S1P, and furthermore PTX and dibutyryl cAMP treatment hardly affected the LPA-induced Ca2+ mobilization. We conclude that exogenous S1P mobilizes Ca2+ through phospholipase C activation. The S1P-induced enzyme activation is at least partly mediated by PTX-sensitive GTP-binding protein-coupled receptors which may be different from LPA receptors.

Adhesiveness for extracellular matrices and lysosomal enzyme release from normal and beta 2 integrin-deficient bovine neutrophils.

The adhesiveness of control and CD18-deficient bovine neutrophils on culture plates precoated with collagen I, collagen IV, fibronectin and laminin was measured to evaluate the possible factors for adherence to extracellular matrices. The release of N-acetyl-beta-D-glucosaminidase (NAGase) from control and CD18-deficient neutrophils stimulated with complement receptor type 3 (CR3) or Fc receptor dependent stimuli was also evaluated. The adhesive activities of CD18-deficient neutrophils to collagen I, collagen IV and fibronectin were significantly diminished (P < 0.05); however, similar adhesion to laminin was observed in CD18-deficient neutrophils and control neutrophils. The adhesive activity of control neutrophils on uncoated plates increased 2.5 times (P < 0.05) with the presence of PMA. The mean activities for NAGase release from CD18-deficient neutrophils stimulated with opsonized zymosan and aggregated bovine immunoglobulin G (Agg-IgG) were 46.7 and 82.7% that of the control neutrophils, respectively. The Agg-IgG-induced NAGase release from control and CD18-deficient neutrophils was eliminated by H7, a protein kinase C inhibitor. These results support that an association between CR3 and Fc receptors on neutrophils appears to play an essential role in neutrophil functions.

Characterization of functions of neutrophils from bone marrow of cattle with leukocyte adhesion deficiency.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

Expression and role of adhesion molecule CD18 on bovine neutrophils.

Expression of CD18 on bovine neutrophils in response to stimulation by zymosan activated serum (ZAS) and phorbol myristate acetate (PMA) and the effects of monoclonal antibodies (MAB) recognizing CD18 or bovine neutrophil surface antigens (S2G8 and S5F8G10) on adherence, chemotactic responses and phagocytosis of bovine neutrophils were evaluated. CD18 expression of neutrophils was increased after ZAS and PMA treatment by 12.2 and 54.2% respectively, and were significantly (p < 0.05, p < 0.01) different from those of untreated neutrophils. CD18 expression by neutrophils from a Holstein-Friesian heifer affected with leukocyte adhesion deficiency was within negative controls when stimulated by ZAS and PMA. Adherence, chemotactic responses, and phagocytosis were significantly decreased (p < 0.01) in neutrophils continuously treated with anti-CD18 MAB (MHM 23). Adherence was also significantly decreased in anti-CD18 pretreated neutrophils. Significant (p < 0.01) differences of chemotactic responses and phagocytosis of neutrophils were found between neutrophils pretreated and continuously treated with anti-CD18 MAB (MHM 23). Monoclonal antibodies to other surface antigens did not significantly alter neutrophil adherence, chemotaxis or phagocytosis. This study demonstrated that CD18 expression on bovine neutrophils is increased significantly by stimulation with ZAS and PMA and that the adhesion molecule CD18 plays an important role in adhesion-related functions.

Enhanced expression of Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P < 0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 micrograms/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).

Proteinuric potentials of angiotensin II, [des-Asp1]-angiotensin II, and [des-Asp1, des-Arg2]-angiotensin II in rats.

To compare proteinuric potentials among angiotensin II (ANG II) and its fragments, [des-Asp1]-angiotensin II (ANG III) and [des-Asp1, des-Arg2]-angiotensin II (ANG IV), the peptide was intravenously infused for 30 min at doses of 0.015, 0.05, 0.15, 0.45 and 1.45 nmol/kg body weight/min. The infusion of ANG II and ANG III increased the fractional clearance of albumin in a dose-dependent manner: most extensively for ANG II, and moderately for ANG III. In contrast, the infusion of ANG IV hardly showed any proteinuric action, even at the maximal dose of 1.45 nmol/kg body weight/min. These results denoted that the cleaving of the N-terminal aspartic acid1 from ANG II weakened the proteinuric action in the glomeruli, and the further cleaving of the N-terminal arginine from ANG III led to a complete loss of proteinuric action in the glomeruli.

Analysis of mononuclear cell functions in Holstein cattle with leukocyte adhesion deficiency.

Lymphocyte functions in cattle affected with leukocyte adhesion deficiency (LAD, termed BLAD in cattle) were evaluated by lymphocyte markers, blastogenic response, and immunoglobulin concentrations; mononuclear phagocyte functions were assessed by chemotactic and luminol-dependent chemiluminescent (CL) responses to determine the effects of impaired expression of leukocyte CD18 on mononuclear cell functions. Deficient CD18 expression on lymphocytes and mononuclear phagocytes from cattle with BLAD was clearly detected by use of flow cytometric analysis. There were no significant differences in the population of peanut agglutinin (PNA)-positive and surface immunoglobulin-bearing blood lymphocytes from clinically normal cattle and cattle with BLAD, as determined by flow cytometric analysis. Lymphocytes from cattle with BLAD had strong mitogen-induced blastogenic responses, which were greater than those from controls. Adherence of mononuclear phagocytes from cattle with BLAD was markedly impaired, and their chemotactic responses had diminished values, compared with those of controls. Luminol-dependent CL of mononuclear phagocytes from affected cattle, stimulated by opsonized zymosan, had significantly (P < 0.01) decreased values, compared with those of controls. Concentrations of IgG were markedly increased in serum from cattle with BLAD, compared with those in controls. These results indicated that impaired expression of leukocyte CD18 has marked effects on adhering activity of mononuclear phagocytes, and significantly inhibits CL response of mononuclear phagocytes mediated by inactivated-complement 3b-dependent functions. High selective immunoglobulin concentrations indicated that lymphocytes of B-cell lineage may have normal function.

Dual pertussis toxin-sensitive pathway of zymosan-induced activation in guinea pig macrophages. An anti-CR3 antibody-inhibitable stimulation of phagocytosis and -resistant stimulation of O2- production and arachidonate release.

Complement receptor type 3 (CR3)-mediated cellular responses in guinea pig macrophages were investigated by using zymosan and serum-opsonized zymosan (SOZ) as the multivalent ligand for CR3. The ingestion of zymosan and SOZ was accompanied by O2- generation and arachidonate release. These responses were suppressed by prior exposure of macrophages to pertussis toxin (PT). Opsonization of zymosan gave rise to more than 6-fold activation of the ingestion, whereas the magnitude of either arachidonate release or O2- generation was unchanged. The Fab' fragment of anti-Z-1, a monoclonal antibody specific for the alpha chain of guinea pig CR3, inhibited the ingestion of zymosan by 60% without affecting zymosan-induced arachidonate release and O2- generation. These data suggested that there might be at least two functionally distinct binding sites for zymosan. O2- generation and arachidonate release might be regulated through one site and phagocytosis another. Both sites should be coupled to PT-sensitive GTP binding protein.

Two cases of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) (case report).

Two Holstein calves showing clinical signs such as ulcerative stomatitis, severe gingivitis, periodontitis, loss of teeth and stunted growth, associated with marked neutrophilia, were evaluated by clinicopathologic analysis, neutrophil functions and flow cytometric analysis of CD18 expression on neutrophils. Decreased CL response, chemotaxis, yeast phagocytosis, and deficient CD18 expression of neutrophils from affected animals were demonstrated. Pathological findings involved were ulcerative gingivitis, severe periodontitis, laryngitis, and multiple ulcers in forestomach. This study demonstrates that neutrophil functions are closely associated with impaired iC3b receptor, and these affected animals were diagnosed as bovine leukocyte adhesion deficiency (BLAD).

High-molecular-weight hyaluronic acids inhibit chemotaxis and phagocytosis but not lysosomal enzyme release induced by receptor-mediated stimulations in guinea pig phagocytes.

The effects of high-molecular-weight (HMW) hyaluronic acids (HAs) of 1.9 x 10(6) Da, 8 x 10(5) Da and 3 x 10(5) Da on the receptor-mediated functions of guinea pig peritoneal phagocytes were studied. HMW-HAs of 1.9 x 10(6) Da (HA190) and 8 x 10(5) Da (HA80) effectively inhibited the chemotactic activity of polymorphonuclear leukocytes (PMNs) for formyl-Met-Leu-Phe (fMLP). The degree of inhibition was dose-dependent and the concentrations of HA190 and HA80 required for 50% inhibition were 0.5-1.5 mg/ml and 1.5-2.5 mg/ml, respectively. HMW-HA of 3 x 10(5) Da (HA30) hardly affected the chemotaxis within a concentration range of 0.5-5.0 mg/ml. The phagocytic activities of PMNs and macrophages (Mphis) for serum-opsonized zymosan (SOZ) and polystyrene latex particles were also inhibited by these HAs in a dose- and molecular-weight-dependent manner and HA190 was again the most inhibitory. By contrast, the release of lysosomal enzyme from Mphis stimulated with SOZ was not significantly affected by HMW-HAs at any concentration used. Furthermore, the binding of [3H]fMLP with PMNs and the rosette formation of Mphis with SOZ were not influenced by the presence of HMW-HAs. These findings suggested that the binding of HMW-HAs to the HA receptors on PMNs and M phi s might produce certain intracellular signals which would be responsible for the suppression of the chemotaxis and the phagocytosis but not for the release of lysosomal enzyme. For the generation of such signals, higher-molecular-weight HMW-HAs would be more effective than lower one.

Bovine leukocyte adhesion deficiency in Holstein cattle.

Two Holstein heifers with persistent and recurrent infections including ulcerative gingivitis, periodontitis, pneumonia, loss of teeth and stunted growth associated with marked neutrophilia were evaluated clinically and for neutrophil function, CD18 expression on neutrophils and CD18 genotype analysis by DNA-polymerase chain reaction (PCR) test. Adherence to nylon fibers and phagocytic activity of neutrophils from affected animals were significantly (p < 0.05) impaired as compared with those of controls. Neutrophils from affected heifers had decreased chemiluminescent (CL) responses when stimulated with opsonized zymosan, compared with those of controls. In contrast, neutrophils from affected heifers produced increased CL responses when stimulated with latex beads and phorbol myristate acetate compared with those of controls. The clinical findings, functional leukocyte abnormalities, deficiency in expression of CD18 on neutrophils, and the D128G mutation detected by DNA-PCR testing of affected heifers demonstrated that these heifers have bovine leukocyte adhesion deficiency (BLAD). Although both animals were confirmed to be homozygotes for BLAD by DNA-PCR test, they had differences in clinical, hematological and neutrophil functional characteristics.

Effects of high-molecular-weight hyaluronates on the functions of guinea pig polymorphonuclear leukocytes.

The effect of hyaluronate (HA) with a molecular weight of 1,900,000 (HA190) purified from products of Streptococcus equi on various functions of guinea pig peritoneal polymorphonuclear leukocytes (PMNs) was examined. HA190 effectively inhibited both the chemotactic activity of PMNs for a chemotactic peptide, formyl-Met-Leu-Phe, and the phagocytic activity of the cells for serum-opsonized zymosan (SOZ) in a dose-dependent manner. When the inhibitory activity of the HAs of different molecular weights, including HA190, HA of molecular weight 800,000 (HA80), and HA of molecular weight 300,000 (HA30), was assessed, the HA190 showed the strongest inhibitory activity and HA30 the lowest activity. In contrast, significant inhibition of the superoxide generation by PMNs upon stimulation with SOZ was not observed even in the presence of HA190 and HA80 at the highest concentration used (2.5 mg/mL). This finding indicated that the HAs studied did not prevent the interaction of PMNs with stimuli. From these data it is concluded that the binding of high-molecular-weight HAs to the HA receptors on PMNs may produce intracellular signals that are responsible for suppression of phagocytosis and chemotaxis but not for superoxide generation.

Further studies on guinea pig Z-1 antigen that is involved in phagocytosis of zymosan by macrophages: cell type distribution of the antigen and cross-reactivity of anti-Z-1 with human cR3.

We have previously identified a heterodimer molecule, Z-1, on guinea pig peritoneal macrophages (M phi s) by the newly prepared monoclonal antibody, anti-Z-1, and Z-1 has been assumed to be the complement receptor type three (CR3) in this species. To clarify this assumption, the cell type distribution of the antigen in guinea pig and the cross-reactivity of anti-Z-1 with other species were analyzed. It was demonstrated that Z-1 was expressed on peritoneal M phi s, pulmonary M phi s, peritoneal polymorphonuclear leukocytes (PMN), peripheral neutrophils, and some subpopulations of spleen cells and of bone marrow cells, but not on erythrocytes, circulating lymphocytes, and lymphocytes in both spleen and bone marrow in detectable amounts. Thus the expression of Z-1 seems to be restricted to phagocytes as is CR3 of other species. Furthermore, it was found that anti-Z-1 bound with peripheral neutrophils from human, horse and goat and HL-60 cells differentiated into monocytes. Any cross-reactivity of the antibody was not detected with neutrophils from rabbit, cow, sheep and dog and nondifferentiated HL-60 cells. Human Z-1 was indistinguishable from human CR3, since both were the heterodimer consisting of alpha chain of 170 kDa (pI = 6.6-7.2) noncovalently associated with beta chain of 100 kDa (pI = 5.6-6.7). In addition, human CR3 in detergent-lysate of neutrophils was completely adsorbed with anti-Z-1 F(ab')2- Sepharose. These findings indicate that guinea pig Z-1 shares an antigenic determinant with human CR3. It thus seems to be possible that Z-1 may function as CR3 in guinea pigs.

Characterization of a monoclonal antibody to guinea pig peritoneal macrophages that inhibits phagocytosis of unopsonized zymosan: structural and functional similarities of the antigen to human and mouse CR3.

A monoclonal antibody, anti-Z-1, was established by fusion of spleen cells from mice immunized with guinea pig thioglycollate-induced peritoneal macrophages (TGC-M phi s) with mouse myeloma cells, P3-X63-Ag8-6.5.3. The Fab' fragments of anti-Z-1 bound to almost all of the TGC-M phi s with a high association constant (6.0 +/- 0.8) X 10(8) M-1, and effectively inhibited phagocytic activities of the cells for unopsonized zymosan and serum-treated zymosan. On the contrary, neither the phagocytic activity for rabbit IgG antibody-sensitized sheep erythrocytes nor that for periodate-treated sheep erythrocytes was inhibited by anti-Z-1. Immunoprecipitation analysis revealed that the antigen recognized by anti-Z-1, which was named Z-1 antigen, consists of a polypeptide chain with a molecular weight of 140,000 (alpha chain) noncovalently associated with a polypeptide chain of 95,000 (beta chain). The epitope with which anti-Z-1 reacts was found to be on the alpha chain by Western blotting. Furthermore, it was found that Z-1 antigen solubilized from the cells with nonionic detergent was capable of binding to unopsonized zymosan, suggesting that Z-1 antigen may function as a receptor for zymosan. These findings show the structural and functional similarities of Z-1 molecules on guinea pig peritoneal macrophages to the third complement receptor on human and mouse leukocytes.

Possible involvement of a 95-kDa protein phosphorylation in phorbol 12-myristate 13-acetate-induced suppression of zymosan phagocytosis in guinea pig macrophages.

Recently, we characterized a surface antigen (Z-1) of guinea pig macrophages by monoclonal anti-Z-1 antibody. The Z-1 antigen consists of two different polypeptide chains; alpha (140 kDa) and beta (95 kDa). This antigen is closely correlated with the phagocytic activity of the cells for zymosan and presumably functions as a receptor for zymosan. In the present study, the effect of phorbol 12-myristate 13-acetate (PMA) on the function of Z-1 was examined. Incubation of ortho-[32P]phosphate-labeled macrophages with PMA greatly increased the phosphorylation of the beta subunit of Z-1 but not that of the alpha subunit. Optimal phosphorylation was observed when cells were incubated with 300 ng/ml of PMA for 60-120 min. The PMA-induced phosphorylation was markedly suppressed by treatment of the macrophages with H-7, an inhibitor of protein kinase C. A chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP) also caused phosphorylation of the beta subunit. Unlike PMA, fMLP maximized the phosphorylation within 30 s. Purified Z-1 was an excellent substrate for the exogenously added protein kinase C only in the presence of both Ca2+ and phosphatidylserine. H-7 completely inhibited the in vitro phosphorylation. These data suggest that the beta subunit of Z-1 is phosphorylated by protein kinase C. The phosphorylation of Z-1 by PMA and fMLP coincided with inhibition of zymosan phagocytosis. A linear relationship was obtained between the level of phosphorylation of Z-1 and the degree of inhibition of zymosan phagocytosis induced by PMA. Thus, the results suggest that zymosan uptake is negatively regulated by protein kinase C-mediated phosphorylation of the beta subunit of Z-1.