J. Basic Microbiol. 30 (1990) 5 , 341-347
(Department of Microbiology and Immunology, ‘Department of Biochemistry Institute of Molecular Biology Jagiellonian University and’) Department of Biocybernetics. Technical University AGH, Krakow, Poland)
Anti-staphylococcal serine proteinase and other serum factors in phagocytosis JACEKMIEDZOBRODZKI, MARIAGORKA,ALICJAWASNIOWSKA’, RYSZARD TADEUSIEWICZ’ and ZOFIAPORWIT-BOBR (Rcceiwd 17 April 1989/Accepted 26 October 1989)
The interactions between polymorphonuclear cells (PMN), Stup/~.vlococcussuprophyticus cells and rabbit antibodies against Staphylococcus uureus V 8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocystosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocystosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase y-globulin serum fraction, anti-serine proteinase IgG, nonimmunized rabbit serum or with complement.
The influence of several serum factors has beeri investigated in the study of the mechanism of action of defence against staphylococcal infection. The process of infections due to staphylococci is complex and multifactorial (CLARK and EASMON 1986, GEMMELL and SCHUMACHER-PERDREAU 1986, HENDRICKS et al. 1986, JEIJASZEWICZet al. 1978, SHEAGREN 1988, WADSTROM and ROZGONYI 1986). It causes a defence reaction consisting of various mechanisms, i.e. enhancement of phagocytosis through opsonic (EASMONand CLARK 1987), bacterial activity by activation of complement activity (EASMON and GLYNN1975, EASMONei af.1975, PRYJMA et ~11.1976) and inactivation of excellular bacterial products, OCHALEK et al. 1989). It has been reported previously that an optimal host defence against staphylococci depends on the integrity of the skin and mucous membranes, antibodies against extracellular products, opsonins and phagocytes. It was also revealed that the presence of antibodies in serum remarkably enhanced the phagocytosis process (EASMON and CLARK 1987, MIEDZOBRODZKI e l al. 1987). In our previous study on staphylococcal serine proteinase (SSP) and human polymorphonuclear cells (PMN) we showed that SSP was a chemoattractant for PMN (BARANet al. 1988). We also reported that preincubation of human PMN with serine proteinase may enhance the process of phagocytosis of some staphylococcal strains (MIEDZOBRODZKT et af. 1987, 1988). The aims of the present investigation were to examine the effects of various serum factors against SSP on the human PMN phagocytosis of a Staphylococcus saprophyticus strain isolated from a clinical case. Materials and methods Phagocytes: Human PMN cells were isolated from the venous blood ofnormal volunteers. They were healthy adults who had not been medicated for more than one month. Blood samples were kindly supplied by Dr. S. PAWLISZYN from Blood Bank, University School of Medicine, Krakow, Poland. PMN were isolated in Uppsala, Sweden) gradient ( B ~ Y U 1968). M Remaining erythrocytes were removed by a Ficoll (PHARMACIA, Preliminary results were presented at Symposium “Role of Granulocytes in Immunology”, October 3-5. 1988. Spala. Poland
342
J. MIEDZOBRODZKI e l ul.
hypotonic lysis. Cell viability determined by trypan blue dye exclusion was higher than 95 "6.More than 97 "; of the cells were polymorphonuclear as assessed by morphology of stained cells. Bacterial strains: Stupliy/ococcus U U ~ Y U SV 8 strain was used as a source of serine proteinase. (Gift from Karolinska Institute, Stockholm, Sweden). Stupholjmccus suprophyticusstrain used in the Dr. S. ARVIDSON, phagocytosis assay was isolated from clinical material of a patient admitted to Institute of Microbiology, University School of Medicine, Krakow, Poland. AH strains were maintained on nutrient agar slants (Tryptic Soy Agar, DIFCO)at 4 "C. S. suprophvticus organisms for all studies were grown in Brain Heart Infusion broth (DIFCO)pH 7.0 for 18 h a t 37 "C, centrifuged for 10 min at 3000 r.p.m. and washed twice with a Parker buffer (BIOMED,Lublin. Poland) to make dilutions for further experiments. The spectrophotometer S P E C O L ~ ~ (CARLZEISS,Jena, D.D.R.) i. = 540 nm was used to control the density of bacterial cells which was confirmed by counting colony forming units. et a/. 1970), (gift from Dr. J. POTEMPA) Antibody preparations: Specific antiproteinase IgG (TULKENS and specific antiproteinase ;-globulin serum fraction were obtained from New Zealand white rabbits immunized with SSP. The standard immunodifusion test was performed to control the specificity of obtained antibodies. Pooled normal serum from healthy nonimmunized New Zealand white rabbits was used as control. In all immunodifusion assays the ratio of serum factors and SSP was estimated. The neutralization test of SSP was determinad only for a a I-plasma inhibitor, and maximum of inhibition of azocasein activity of proteinase was in the ration 1 : 10. In this ratio the immunodifusion assay was also positive. Complement : Normal guinea pig serum (Lyophilized complement, BIOMED.Krakow, Poland) was used. Serine proteinase: For immunization of rabbits and for phagocytosis, serine proteinase produced by Sfujhy/ocuccus uureus V 8 strain was isolated and purified according to DRAPBAU (1976, 1978) in our laboratory. The proteolytic activity of SSP was estimated using aszocasein as a substrate. The activity of enzyme was 6.000 units of activity calculated on the basis of absorbance increase of 1 unit activity per 1 mg of protein at 37 "C and 60 min of incubation. Lyophilized SSP had the end activity 4 unitslml pig of protein. Phagocystosis assay: Phagocytosis by PMN was assayed in siliconized tubes basically according to the et ul. 1987). Briefly, equal volumes 0.2 ml of PMN method used in our previous study (MIEDZOBRODZKI suspension at the concentration of 5.5 x 10" cells and staphylococci at the concentration of 3 x 10' bacteria/ mi were introduced into capped tubes. The final bacteria to phagocyte ratio was 55: 1. Additionally, following protcin preparations were added: I. 0.1 ml of IgG specific anti-proteinase, 11. 0.1 ml of y-globulin serum fraction specific antiproteinase, 111. 0.2 ml of normal rabbit serum, and IV. 0.1 ml of standard complement. One set of four presented tests was supplemented with SSP at the concentration of 1.25 pg/ml(5 units) and the next set was enriched with enzyme at the concentration of 12.5 pg/ml (SO units). The third set was assayed without any proteinase as a control. Smears were GIEMSA stained. The number of bacteria in fifty phagocytes in each of six replicates was counted. Phagocytosis index was expressed as a ratio of average number of bacteria to phagocytes. Statistics: The basis of the applied method was a computer aided calculation of the linear and logarithmic correlation coefficients between phagocytosis and specific or normal serum factors presented in the assays. These coefficients were compared with their critical values calculated from the t-student method at the given level of significane (TADEUSIEWICZ 1986). All the experimental data and calculations were stored in the result matrix. Statistically significant elements of the matrix were used to evaluate parameters of linear regression between proteins added to the samples and the phagocytosis. This type of algorithm was repeated for each .type of protein, i.e. IgG. globulin, normal serum or complement.
Results
The assay values obtained from phagocytosis were as follows : the first observation reported in Fig. I supports concept that the complement enhances the phagocytosis process. The presence of the complement in the control test considerably stimulated the phagocytosis of S. suprophyticus cells by human PMN cells (according to the previous report of EASMON and CLARK1987). An inhibition of the phagocytosis process caused by presence of SSP in the test with the complement was observed. The anti-serine proteinase antibodies i.e. specific IgG and y-globulin serum fraction formed antigen-antibody complexes that enhanced phagocytosis process by opsonization of S. suprophyticus cells. (Fig. 2, Fig. 3). The normal rabbit serum from nonirnmunized animals enhanced the phagocytosis of S. suproplq~timscells due to human PMN cells, (Fig. 4).
Anti-proteinase factors and phagocytosis
no complement, no
343
SSP
U SSP conc. Opg/rn/ E2 SSP conc. f.ZSpg/m/ 69 SSP conc. f2.5,ug/m/
Fig. 1 The effect of proteinase and complement on phagocytosis. PMN cells and bacteria were incubated with proteinase (5 units of 50 units) or with a PARKERmedium or complement. The process of phagocytosis was stopped in the ice bath. Smears were stained in GIEMSA.
Discussion As was reported in our previous study, the phagocytosis of S. saprophyticus due to human polymorphonuclear cells was inhibited by low and enhanced by higher concentrations of S. aureus V 8 serine proteinase (MIEDZOBRODZKI et al. 1987, 1988). A limited proteolysis caused by SSP modifies protein structures on phagocytic cell surface (GIRMAet al. 1986), TARCOWSKI and NIEMIALTOWSKI 1986). As demonstrated by PROKESOVA el al. (1988) SSP has no cytotoxic effect on human PMN cells over a broad concentration range. The presence of SSP in the host causes interactions with proteinase inhibitors but a part of the enzyme can also modify surface proteins of the host cells or proteins of other organisms accompanying staphylococci in mixed infections with various microorganisms (CIBOROWSKI and JELJASZEWICZ 1985, JONESand FISCHETTI 1988, TASHIROet al. 1987). In the case of nonstaphylococcal bacteria after the end of a previous staphylococcal infection, a trace of serine proteinase may also change the protein surface receptors of secondary intruders in host (BEVER and WHITAKER 1985, SHEAGREN 1988). The interactions between SSP, anti-serine proteinase antibodies, nonspecific serum fraction proteins and surface proteins of bacterial cells and phagocytes compose many complex reactions, which influence the process of phagocytosis. It is necessary to stress the role of opsonization caused by specific or nonspecific opsonis and increasing the process of phagocytosis. S. saprophyticus cells were opsonized better by complexes formed by IgG, y-globulin or nonspecific proteins with SSP than by such single proteins as IgG, y-globulin or normal serum nonspecific opsonins. This interpretation may be -tentatively
344
J. MIEDZOBRODZKI e l a/.
Fig. 2 The effect of proteinase and antiproteinase IgG on phagocytosis. PMN cells and bacteria were incubated with rabbit-specific antiproteinase IgG and with proteinase ( 5 units or 50 units) or with a PARKER medium.
used for an explanation of higher values of phagocytosis index in the assays under consideration (OCHALEK e t a / . 1989). That the highest values of phagocytosis index were observed in the presence of normal serum is of special interest. It seems that the cr-2 macroglobulin serum fraction known as a serine proteinase inhibitor, is responsible for the formation of SSP-antiproteinase-complexes. Probably conformationally changed complexes stimulate opsonization and enhance the phagocytosis process. a-2 macroglobulin is one of the major antiproteinases found in the plasma of vertebrates (BRETAUDIERE et a/. 1988, DUBINet a/. 1984 and FELDMAN et al. 1985). Multimolecular complexes might be formed and opsonized bacteria, enhancing phagocytosis. For both does of SSP: 5 units and 50 units, a stimulation was observed in the phagocytosis tests. However, higher phagocytosis index values were noted for the lower concentration of SSP. The inhibitory activity of serum factors was not tested, but the protein concentrations in all samples were similar. The main plasma inhibitors (a 1-PI and other serpins) are modified after incubation with SSP (to be published), however, cr-2 macroglobulin molecules can neutralise SSP. Generally, serum was found t o protect the proteinase treated lymphocytes (PORWIT-BOBR et al. 1989). At the higher concentration of SSP the excess of the non-bound enzyme could impair phagocytic cells by a modification of surface receptor proteins, therefore values of phagocytosis index were lower than in assays made with the lower concentration of proteinase. Finally, phagocytosis of S. saprophyticus cells that had been treated with anti-serine proteinase antibodies was markedly increased but the normal rabbit serum also caused a similar effect. Of interest are further investigations analysing the cross reaction between
345
Anti-proteinase factors and phagocytosis
Fig. 3 The effect of proteinase and antiproteinase y-globulin on phagocytosis. PMN cell and bacteria were incubated with rabbit-specific antiproteinase y-globulin and with proteinase (5 units or 50 units) or with a PARKER medium.
30
25
0 55Pconc. Opg/ml El SSP conc. 1.25pg/ml
6Y SSP conc.
t2.5pg/ml
‘c
i%
-s 2c .2
.G a
5 15 0,
K
5
C Fig. 4 The effect of proteinase and normal serum on phagocytosis. PMN cells and bacteria were incubated with normal rabbit serum and with proteinase ( 5 units or 50 units) or with a PARKERmedium.
346
J. MlEDZOBRODZKl e f al.
different proteins and their influence on phagocytosis in septicaemia or common case of short physiological bacteremias due to proteinase positive S. aureus strains together with coagulase-negative staphylococci or accompanying microorganisms normally present on skin and mucous membranes. Acknowledgements We wish to thank Mrs. DANUTA SZMYDfor her excellent technical assistance. This investigation was supported in part by Grant CPBP 04. 02. 1. 3. 1. from the Polish Ministry of Education.
References BARAN,K., MIEDZOBRODZKI, and PORWIT-BOBR, Z., 1988. Preliminary estimation of chemoattractant activity of staphylococcal serine proteinase in vitro. Ant. Leeuwenhoek J. Microbiol., 54, 85-87. BRETAUDIERE, J. P., TAPON-BRETAUDIERE, J. and STOOPS,J. K., 1988. Structure of native a-2-macroglobulin and its transformation to the protease bound form. Proc. Natl. Acad. Sci. USA, 85. 1437-1441. BEVER.C. T. and WHITAKER, J. N., 1985. Proteinase in inflammatory demyelinating disease. Springer Semin. Imrnunopathol., 8. 235-240. BOYUM, A., 1968. Separation of leukocytes from blood and bone marrow. Scand. J. Clm. Lab. Invest., 21, 77-80. CIBOROWSKI, P. and JEUASZEWICZ, J., 1985. Staphylococcal enzymes and virulence. In: Bacterial Enzymes and Virulence (I. A. HOLI)ER, Editor), pp. 145-183. CRC Press, Boca Raton, FL. CLARK, L. A. and EASMON. C. S. F., 1986. Opsonic requirements of Staphylococcus epiclermidis. J. Med. Microbiol., 22, 1-9. DRAPEAU, G. R., 1976. Protease from Staphvlococcus aureus. Methods Enzymol., 45, 469-475. G. R., 1978. The primary structure of staphylococcal protease. Can. J . Biochem., 56, 534-544. DRAPEAU, DUBIN,A,, POTEMPA, J . and SILBERRING, J., 1984. a-2-macroglobulin from horse plasma. Purification, properties and interaction with certain serine proteinases. Biochem. Intern., 8, 589-596. EASMIN. C. S. F. and CLARK, L. A,. 1987. Opsonization of Staphylococcus epidermidis. Zbl. Bakt. Suppl., 16. 169-176. EASMON, C. S. F. and GLY“, A. A,, 1975. The role of humoral immunity and acute inflammation in protection against staphylococcal dermonecrosis. Immunology, 29, 67-74. EASMON, C. S. F., HAMILTON, 1. and GLYNN, A. A., 1975. Mode action of a staphylococcal antiinflammatory factor. Br. J. Exp. Pathol., 54, 638-645. FELDMAN, S. R., GONIAS, S. L. and PIZZO,S. V., 1985. Model of a-2-macroglobulin structure and function. Proc. Natl. Acad. Sci. USA, 82, 5700-5704. C. G. and SCHUMACHER-PERDREAU, F.. 1986. Extracellular toxins and enzymes elaborayed by GEMMELL, coagulase-negative staphylococci. In : Coagulase-Negative Staphylococci (P. A. MARDH,K. H. SCHLEIFER, Editors), pp. Almqvist-Wiksell Stockholm. GIRMA, J. P., CHOPEK, M. W., TITANI,K. and DAME,E. W., 1986. Limited proteolysis of human von WILLEBRAND Factor by Staphylococcus aureus V 8 protease: Isolation and partial characterization of a plateletbinding domain. Biochemistry, 25. 3156-3163. HENDRICKS, P. A. J., VAN DER TOL,M. E., ENGELS, F., NIJKAMP, F. P. and VERHOEF, J., 1986. Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis of Staphylococcus aureus. Inflammation, 10, 37-42. J ., SZMIGIELSKI, S. and HRYNIEWICZ, w . , 1978. Biological effects of stapholococcal and JEUASZEWICZ, and T. WADSTROM, streptococcal toxins. In: Bacterial Toxins and Cell Membranes (J. JELJASZEWICZ Editors), pp. 185-228. Academic Press London New York. JONES, K. F. and FISCHETTI, V. A,, 1988. The importaqce of the location of antibody binding on the M 6 protein for opsonization and phagocytosis of group A M 6 streptococci. J . Exp. Med., 167, 1114-1123. J., GORKA,M. and PORWIT-BOBR, Z., 1988. Phagocytic activity of human granulocytes MIEDZOBRODZKI, stimulated by staphylococcal serine proteinase. In: Polish, Symposium: “Role of granulpcytes in immunology”. Oct. 3-5, Spala, Poland, Materials, Abstract Nr. 30.
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MIEDZOBRODZKI, J., TADEUSIEWICZ, R. and PORWIT-BOBR, Z., 1987. Evaluation of the effect of staphylococcal serine proteinase on phagocytosis. Arch. Immunol. Ther. Exp., 35,877 -885. OCHALEK, T.. TURYNA. B. and PORWIT-BOBR, Z., 1989. Mechanism of staphylococcal serine proteinase inactivation by lymphocytes and granulocytes. Anton Leeuwenhoek J . Microbiol.. -in press. T., PROKESOVA, L., JOHN,C., BARAN, K. and POTEMPA, J., 1989. Effects of PORWIT-BOBR, Z., OCHALEK, serine proteinase from Staphylococcus aureus V8 cells on con A stimulation of human lymphocytes. Microbiologica, 12, 139- 142. PROKESOVA, L., PORWIT-BOER, Z., BARAN, Z., POTEMPA, J. and JOHN,C., 1988. Effect of serine proteinase from S/apl~ylococcusaureus on in uitro stimulation of human lymphocytes. Immunol. Lett., 19, 127- 132. J . , PRYJMA, K., GROV,A. and HECZKO,P. B., 1976. Immunological activity of staphylococcal PRYJMA, Editor), pp. 875-881. cell wall antigens. In: Staphylococci and Staphylococcal Diseases (J. JEUASZEWICZ, Fischer Stuttgart New York. SHEAGREN, J. N., 1988. Inflammation induced by Srap/iyIoco~cu~ aureus. In : Inflammation Basic Principles and R. SNYDERMAN, Editors), pp. 829-840. and Clinical Correlates (J. I. GALLIN,I. M. GOLDSTEIN Raven Press Ltd. New York. SMITH,R. J., SPEZIALE, S. C., SM, L. M. and BOWMAN, B. J., 1986. Requirement for cell-bound proteases in the mechanism of human neutrophil activation with various stimuli. Inflammation, 10, 257-262. TADEUSIEWICZ, R., 1986. Methods and principles of application of the digital computers for elaboration biological experiments. In Polish.. Prace Naukowe Instytutu Cybernetyki Politechniki Wroclawskiej, S. Konferencje, 41, 239-244. S. P. and NIEMIALTOWSKI, M., 1986. Appearance of Fc receptors on polymorphonuclear leuTARGOWSKI, kocytes after migration and their role in phagocytosis. Infec. Immunity, 52, 798-803. TASHIRO,M., CIBOROWSKI, P., REINACHER, M., PULVERER, G., KLENK,H. D. and ROTT,R., 1987. Synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity. Virology, 157, 421-430. TULKENS, P., TRONET,A. and HOOF,F., 1970. Immunological inhibiting of lysosome function. Nature, 228, 1282- 1284. WADSTROM, T. and ROZGONYI, F., 1986. Virulence determinants of coagulase-negative staphylococci. In: Editors), pp. 123-130, AlmCoagulase-Negative Staphylococci (P. A. MARDHand K. H. SCHLEIFER, qvist-Wiksell Stockholm. Mailing address: Dr. J. MIEDZOBRODZKI, Department of Microbiology and Immunology, Institute of Molecular Biology, Jagiellonian University, Al. Miciewicza 3, 31-120 Krakow, Poland
(Department of Microbiology and Immunology, ‘Department of Biochemistry Institute of Molecular Biology Jagiellonian University and’) Department of Biocybernetics. Technical University AGH, Krakow, Poland)
Anti-staphylococcal serine proteinase and other serum factors in phagocytosis JACEKMIEDZOBRODZKI, MARIAGORKA,ALICJAWASNIOWSKA’, RYSZARD TADEUSIEWICZ’ and ZOFIAPORWIT-BOBR (Rcceiwd 17 April 1989/Accepted 26 October 1989)
The interactions between polymorphonuclear cells (PMN), Stup/~.vlococcussuprophyticus cells and rabbit antibodies against Staphylococcus uureus V 8 serine proteinase or normal rabbit serum proteins were investigated. The effect of opsonization on phagocystosis due to human peripheral polymorphonuclear cells was measured. The results were as follows: phagocystosis index values were relatively increased after the incubation of PMN cells with anti-serine proteinase y-globulin serum fraction, anti-serine proteinase IgG, nonimmunized rabbit serum or with complement.
The influence of several serum factors has beeri investigated in the study of the mechanism of action of defence against staphylococcal infection. The process of infections due to staphylococci is complex and multifactorial (CLARK and EASMON 1986, GEMMELL and SCHUMACHER-PERDREAU 1986, HENDRICKS et al. 1986, JEIJASZEWICZet al. 1978, SHEAGREN 1988, WADSTROM and ROZGONYI 1986). It causes a defence reaction consisting of various mechanisms, i.e. enhancement of phagocytosis through opsonic (EASMONand CLARK 1987), bacterial activity by activation of complement activity (EASMON and GLYNN1975, EASMONei af.1975, PRYJMA et ~11.1976) and inactivation of excellular bacterial products, OCHALEK et al. 1989). It has been reported previously that an optimal host defence against staphylococci depends on the integrity of the skin and mucous membranes, antibodies against extracellular products, opsonins and phagocytes. It was also revealed that the presence of antibodies in serum remarkably enhanced the phagocytosis process (EASMON and CLARK 1987, MIEDZOBRODZKI e l al. 1987). In our previous study on staphylococcal serine proteinase (SSP) and human polymorphonuclear cells (PMN) we showed that SSP was a chemoattractant for PMN (BARANet al. 1988). We also reported that preincubation of human PMN with serine proteinase may enhance the process of phagocytosis of some staphylococcal strains (MIEDZOBRODZKT et af. 1987, 1988). The aims of the present investigation were to examine the effects of various serum factors against SSP on the human PMN phagocytosis of a Staphylococcus saprophyticus strain isolated from a clinical case. Materials and methods Phagocytes: Human PMN cells were isolated from the venous blood ofnormal volunteers. They were healthy adults who had not been medicated for more than one month. Blood samples were kindly supplied by Dr. S. PAWLISZYN from Blood Bank, University School of Medicine, Krakow, Poland. PMN were isolated in Uppsala, Sweden) gradient ( B ~ Y U 1968). M Remaining erythrocytes were removed by a Ficoll (PHARMACIA, Preliminary results were presented at Symposium “Role of Granulocytes in Immunology”, October 3-5. 1988. Spala. Poland
342
J. MIEDZOBRODZKI e l ul.
hypotonic lysis. Cell viability determined by trypan blue dye exclusion was higher than 95 "6.More than 97 "; of the cells were polymorphonuclear as assessed by morphology of stained cells. Bacterial strains: Stupliy/ococcus U U ~ Y U SV 8 strain was used as a source of serine proteinase. (Gift from Karolinska Institute, Stockholm, Sweden). Stupholjmccus suprophyticusstrain used in the Dr. S. ARVIDSON, phagocytosis assay was isolated from clinical material of a patient admitted to Institute of Microbiology, University School of Medicine, Krakow, Poland. AH strains were maintained on nutrient agar slants (Tryptic Soy Agar, DIFCO)at 4 "C. S. suprophvticus organisms for all studies were grown in Brain Heart Infusion broth (DIFCO)pH 7.0 for 18 h a t 37 "C, centrifuged for 10 min at 3000 r.p.m. and washed twice with a Parker buffer (BIOMED,Lublin. Poland) to make dilutions for further experiments. The spectrophotometer S P E C O L ~ ~ (CARLZEISS,Jena, D.D.R.) i. = 540 nm was used to control the density of bacterial cells which was confirmed by counting colony forming units. et a/. 1970), (gift from Dr. J. POTEMPA) Antibody preparations: Specific antiproteinase IgG (TULKENS and specific antiproteinase ;-globulin serum fraction were obtained from New Zealand white rabbits immunized with SSP. The standard immunodifusion test was performed to control the specificity of obtained antibodies. Pooled normal serum from healthy nonimmunized New Zealand white rabbits was used as control. In all immunodifusion assays the ratio of serum factors and SSP was estimated. The neutralization test of SSP was determinad only for a a I-plasma inhibitor, and maximum of inhibition of azocasein activity of proteinase was in the ration 1 : 10. In this ratio the immunodifusion assay was also positive. Complement : Normal guinea pig serum (Lyophilized complement, BIOMED.Krakow, Poland) was used. Serine proteinase: For immunization of rabbits and for phagocytosis, serine proteinase produced by Sfujhy/ocuccus uureus V 8 strain was isolated and purified according to DRAPBAU (1976, 1978) in our laboratory. The proteolytic activity of SSP was estimated using aszocasein as a substrate. The activity of enzyme was 6.000 units of activity calculated on the basis of absorbance increase of 1 unit activity per 1 mg of protein at 37 "C and 60 min of incubation. Lyophilized SSP had the end activity 4 unitslml pig of protein. Phagocystosis assay: Phagocytosis by PMN was assayed in siliconized tubes basically according to the et ul. 1987). Briefly, equal volumes 0.2 ml of PMN method used in our previous study (MIEDZOBRODZKI suspension at the concentration of 5.5 x 10" cells and staphylococci at the concentration of 3 x 10' bacteria/ mi were introduced into capped tubes. The final bacteria to phagocyte ratio was 55: 1. Additionally, following protcin preparations were added: I. 0.1 ml of IgG specific anti-proteinase, 11. 0.1 ml of y-globulin serum fraction specific antiproteinase, 111. 0.2 ml of normal rabbit serum, and IV. 0.1 ml of standard complement. One set of four presented tests was supplemented with SSP at the concentration of 1.25 pg/ml(5 units) and the next set was enriched with enzyme at the concentration of 12.5 pg/ml (SO units). The third set was assayed without any proteinase as a control. Smears were GIEMSA stained. The number of bacteria in fifty phagocytes in each of six replicates was counted. Phagocytosis index was expressed as a ratio of average number of bacteria to phagocytes. Statistics: The basis of the applied method was a computer aided calculation of the linear and logarithmic correlation coefficients between phagocytosis and specific or normal serum factors presented in the assays. These coefficients were compared with their critical values calculated from the t-student method at the given level of significane (TADEUSIEWICZ 1986). All the experimental data and calculations were stored in the result matrix. Statistically significant elements of the matrix were used to evaluate parameters of linear regression between proteins added to the samples and the phagocytosis. This type of algorithm was repeated for each .type of protein, i.e. IgG. globulin, normal serum or complement.
Results
The assay values obtained from phagocytosis were as follows : the first observation reported in Fig. I supports concept that the complement enhances the phagocytosis process. The presence of the complement in the control test considerably stimulated the phagocytosis of S. suprophyticus cells by human PMN cells (according to the previous report of EASMON and CLARK1987). An inhibition of the phagocytosis process caused by presence of SSP in the test with the complement was observed. The anti-serine proteinase antibodies i.e. specific IgG and y-globulin serum fraction formed antigen-antibody complexes that enhanced phagocytosis process by opsonization of S. suprophyticus cells. (Fig. 2, Fig. 3). The normal rabbit serum from nonirnmunized animals enhanced the phagocytosis of S. suproplq~timscells due to human PMN cells, (Fig. 4).
Anti-proteinase factors and phagocytosis
no complement, no
343
SSP
U SSP conc. Opg/rn/ E2 SSP conc. f.ZSpg/m/ 69 SSP conc. f2.5,ug/m/
Fig. 1 The effect of proteinase and complement on phagocytosis. PMN cells and bacteria were incubated with proteinase (5 units of 50 units) or with a PARKERmedium or complement. The process of phagocytosis was stopped in the ice bath. Smears were stained in GIEMSA.
Discussion As was reported in our previous study, the phagocytosis of S. saprophyticus due to human polymorphonuclear cells was inhibited by low and enhanced by higher concentrations of S. aureus V 8 serine proteinase (MIEDZOBRODZKI et al. 1987, 1988). A limited proteolysis caused by SSP modifies protein structures on phagocytic cell surface (GIRMAet al. 1986), TARCOWSKI and NIEMIALTOWSKI 1986). As demonstrated by PROKESOVA el al. (1988) SSP has no cytotoxic effect on human PMN cells over a broad concentration range. The presence of SSP in the host causes interactions with proteinase inhibitors but a part of the enzyme can also modify surface proteins of the host cells or proteins of other organisms accompanying staphylococci in mixed infections with various microorganisms (CIBOROWSKI and JELJASZEWICZ 1985, JONESand FISCHETTI 1988, TASHIROet al. 1987). In the case of nonstaphylococcal bacteria after the end of a previous staphylococcal infection, a trace of serine proteinase may also change the protein surface receptors of secondary intruders in host (BEVER and WHITAKER 1985, SHEAGREN 1988). The interactions between SSP, anti-serine proteinase antibodies, nonspecific serum fraction proteins and surface proteins of bacterial cells and phagocytes compose many complex reactions, which influence the process of phagocytosis. It is necessary to stress the role of opsonization caused by specific or nonspecific opsonis and increasing the process of phagocytosis. S. saprophyticus cells were opsonized better by complexes formed by IgG, y-globulin or nonspecific proteins with SSP than by such single proteins as IgG, y-globulin or normal serum nonspecific opsonins. This interpretation may be -tentatively
344
J. MIEDZOBRODZKI e l a/.
Fig. 2 The effect of proteinase and antiproteinase IgG on phagocytosis. PMN cells and bacteria were incubated with rabbit-specific antiproteinase IgG and with proteinase ( 5 units or 50 units) or with a PARKER medium.
used for an explanation of higher values of phagocytosis index in the assays under consideration (OCHALEK e t a / . 1989). That the highest values of phagocytosis index were observed in the presence of normal serum is of special interest. It seems that the cr-2 macroglobulin serum fraction known as a serine proteinase inhibitor, is responsible for the formation of SSP-antiproteinase-complexes. Probably conformationally changed complexes stimulate opsonization and enhance the phagocytosis process. a-2 macroglobulin is one of the major antiproteinases found in the plasma of vertebrates (BRETAUDIERE et a/. 1988, DUBINet a/. 1984 and FELDMAN et al. 1985). Multimolecular complexes might be formed and opsonized bacteria, enhancing phagocytosis. For both does of SSP: 5 units and 50 units, a stimulation was observed in the phagocytosis tests. However, higher phagocytosis index values were noted for the lower concentration of SSP. The inhibitory activity of serum factors was not tested, but the protein concentrations in all samples were similar. The main plasma inhibitors (a 1-PI and other serpins) are modified after incubation with SSP (to be published), however, cr-2 macroglobulin molecules can neutralise SSP. Generally, serum was found t o protect the proteinase treated lymphocytes (PORWIT-BOBR et al. 1989). At the higher concentration of SSP the excess of the non-bound enzyme could impair phagocytic cells by a modification of surface receptor proteins, therefore values of phagocytosis index were lower than in assays made with the lower concentration of proteinase. Finally, phagocytosis of S. saprophyticus cells that had been treated with anti-serine proteinase antibodies was markedly increased but the normal rabbit serum also caused a similar effect. Of interest are further investigations analysing the cross reaction between
345
Anti-proteinase factors and phagocytosis
Fig. 3 The effect of proteinase and antiproteinase y-globulin on phagocytosis. PMN cell and bacteria were incubated with rabbit-specific antiproteinase y-globulin and with proteinase (5 units or 50 units) or with a PARKER medium.
30
25
0 55Pconc. Opg/ml El SSP conc. 1.25pg/ml
6Y SSP conc.
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i%
-s 2c .2
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K
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C Fig. 4 The effect of proteinase and normal serum on phagocytosis. PMN cells and bacteria were incubated with normal rabbit serum and with proteinase ( 5 units or 50 units) or with a PARKERmedium.
346
J. MlEDZOBRODZKl e f al.
different proteins and their influence on phagocytosis in septicaemia or common case of short physiological bacteremias due to proteinase positive S. aureus strains together with coagulase-negative staphylococci or accompanying microorganisms normally present on skin and mucous membranes. Acknowledgements We wish to thank Mrs. DANUTA SZMYDfor her excellent technical assistance. This investigation was supported in part by Grant CPBP 04. 02. 1. 3. 1. from the Polish Ministry of Education.
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