Angiotensin II augmentation of tyrosine kinase activity in human adherent mononuclear cells

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

45, 48-55 (1991)

Angiotensin II Augmentation of Tyrosine Kinase Activity in Human Adherent Mononuclear Cells’ MICHAEL

R. SIMON,“?

MITCHELL T. KAMLAY,? SUDHIR G. DESAI,* ADHIP P. N. MAJUMDAR*‘t,$

AND

*Medical and TResearch Services, VA Medical Center, Allen Park, Michigan 48101, and the *Department of Medicine, Wayne State University School of Medicine, and *Department of Nurrition and Food Science, Wayne State University, Detroit, Michigan

Received May 8, 1990 and in revised form July 3, 1990

The activity of angiotensin converting enzyme is elevated in the serum of patients with active sarcoidosis as compared with that of healthy persons (1). Angiotensin converting enzyme is present in epithelioid cells of sarcoid granulomas (2), and in blood monocytes from patients with sarcoidosis (3). It is also present in alveolar macrophages from healthy individuals (4,s). The components of the angiotensin system have been identified in alveolar macrophages from healthy mice (6,7) as well as in murine schistosome granuloma macrophages (8). Angiotensin II, a product of angiotensin converting enzyme, is biologically active (9,IO).

The relationship of angiotensin converting enzyme activity and angiotensin II to the inflammatory process in diseases such as sarcoidosis remains unclear. We formulated the hypothesis that granuloma macrophages regulate inflammation by release of angiotensin converting enzyme, which produces angiotensin II, and that angiotensin II in turn modulates monocyte/macrophage activity. Since tyrosine kinase catalyzes phosphorylation of tyrosine residues in proteins and is important in signal transduction and cellular activation (1 l), we further postulated that monocyte tyrosine kinases play a role in the regulation of this process. To address this question, we studied the effect of angiotensin II on mononuclear cell tyrosine kinase activity and tyrosine phosphorylation of proteins in adherent mononuclear cells from healthy individuals. METHODS Adherent Mononuclear Cell Preparation and Culture

A mononuclear cell suspension was obtained from preservative-free heparinized (Sigma Chemical Co., St. Louis, MO) blood by Ficoll-Hypaque (Lympho’ Supported by the Medical Research Service of the Department of Veterans Affairs. 48 0885-4505/91 $3.00 Copyright All rights

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

AI1 AUGMENTATION

OF TYROSINE

KINASE

49

cyte Separation Medium, Organon Teknika, Durham, NC) density-gradient centrifugation. The cells were washed twice in Hanks’ balanced salt solution (HBSS). The cell suspensions at 22°C are adjusted to 2 x lo6 viable mononuclear cells/ml with 20% autologous plasma and 80% RPM1 1640 medium (100 U/ml penicillin, 100 pg/ml streptomycin, and 2 mM glutamine) (all from Gibco, Grand Island, NY). Three-milliliter aliquots containing 6 X lo6 mononuclear cells are placed in flat-bottomed 35 x 14-mm wells (MultiWell, Falcon Plastics, Oxnard, Ca). After 2 hr adherence (37°C 5% COz, 100% humidity), the adherent cell monolayers are washed twice with 37°C RPM1 1640 medium, and then overlaid with 20% autologous plasma and 80% RPM1 1640 containing penicillin, streptomycin, and glutamine, as above. During the first 24 hr of culture, the monocytes were exposed either to no angiotensin II or to 10e5 M angiotensin II. Two subjects’ adherent mononuclear cells were exposed to concentrations of angiotensin II ranging from lo-l3 to 1O-4 M angiotensin II. During the last 24 hr of culture in fresh medium the adherent monocytes either were not stimulated or were stimulated with 10 pg/ml lipopolysaccharide B (LPS, Escherichia coli 026: B6; Difco, Detroit, MI) for 24 hr. The adherent mononuclear cells were removed from the plastic wells by gently scraping them with a rubber policeman. Adherent

Mononuclear

Cell Tyrosine

Kinase Activity

Cellular tyrosine kinase activity was determined as previously described (12). A cell pellet was prepared from adherent mononuclear cells. The pellet was resuspended in 200 ~1 of 10 mM Hepes buffer containing 150 mM NaCl, 1 mM MgClz , 1 mM Na3V04, and 0.1% Triton-X 100. Fifteen microliters of resuspended pellet was added to 35 ~1 of the reaction mixture which in its final volume contained 20 pg poly (L-Glu-L-Tyr; 4: 1) (Sigma, St. Louis, MO) and 2.5 kmol Tris-HCl, 2.5 kmol MgC&, 0.5 nmol Na,VO,, 3.0 pmole adenosine triphosphate (ATP), and 0.4 PCi [y-3’P]ATP (11.7 Ci/mmole; New England Nuclear, Boston, MA). Orthovanadate inhibits degradation of ATP and dephosphorylation of phosphorylated peptides. The reaction proceeded for 10 min at 24°C and was stopped by spotting 20 ~1 of the reaction mixture on 1.5 cm2 of No. 3MM Whatman filter paper. The filter paper was washed twice with 10% trichloroacetic acid containing 10 mM sodium pyrophosphate and allowed to stand for 18 hr at 4°C. It was then rinsed with 95% ethanol before being dried and counted using the 14C channel of a Packard Tri-Carb liquid scintillation counter. Results are expressed as picomoles of 32P incorporated per milligram of protein. Protein was assayed using the method of Bradford (Bio-Rad Protein Assay, Bio-Rad, Richmond, CA) (13). Autophosphorylation of Phosphorylated

and Molecular Proteins

Weight Determination

The approximate molecular weight of the tyrosyl-phosphorylated proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Autophosphorylation and identification of tyrosyl-phosphorylated proteins were accomplished as described by Majumdar and Arlow (14). The 50-~1 reaction mixture contained 2.5 mmole Hepes (pH 7.8), 2.5 mmole MgCl,, 0.5

50

SIMON

ET AL.

nmole orthovanadate, 0.5 nmole [32P]ATP (4 x lo6 dpm), and 0.02% Triton X100. The reaction was initiated with the addition of 15 to 20 pug/ml protein in lysates from adherent cells and proceeded at 4°C for 30 min. The reaction was terminated by the addition of an equal volume of 100°C buffer containing 62.5 mM Tris-HCI (pH 6.8)/6% SDS/20% glycerol/lO% P-mercaptoethanol. The sample was then electrophoresed on a 7.5% polyacrylamide slab gel (1.5 mm) containing 0.1% SDS. After electrophoresis, the gels were fixed for 12 hr in fixing buffer containing 50% methanol, 10% acetic acid, and 40% water. They were then washed in water, and incubated in 1 M KOH at 56°C for 2 hr to destroy alkali-sensitive phosphoserine or phosphothreonine bonds (15,16). The gel was then washed again with water, fixed for 12 hr in 10% acetic acid- 10% isopropanol, dried, and finally exposed to Kodak X-Omat AR film for 3-4 days at -70°C. The molecular weights of the 32P-phosphorylated proteins designated by the labeled bands were calculated from concurrently run marker proteins of known molecular weight (Pharmacia Fine Chemicals, Piscataway, NJ). Subjects

Subjects were I1 self-selected healthy volunteer hospital workers and medical students. Eight were men and three were women (mean age = 27.7 years, SD = 6.6 years, range = 22-40 years). All subjectgs were paid for their participation. RESULTS Adherent

Mononuclear

Cell Tyrosine

Kinase Activity

Experiments were performed in which tyrosine activity was measured in LPSstimulated monocytes which had previously been exposed to angiotensin II. Angiotensin II at 1O-5 M caused significant increases in tyrosine kinase activity in 5 of 11 healthy subjects (Tables 1 and 2). Two of the “responder” subjects’ mononuclear cells demonstrated an angiotensin II concentration dependence effect upon the augmentation of tyrosine kinase activity (Fig. 1). The maximal stimulation of tyrosine kinase varied from 3 1 to 506% and was achieved following incubation of cells with lo-’ M angiotensin II (Table 1). Adherent mononuclear cells which were exposed to angiotensin II but not stimulated with LPS manifested variable effects upon tyrosine kinase activity (Table 1). The mean basal levels of tyrosine kinase activity of the “responder” and “nonresponder” subjects were not different [563 _+ 184 (SE) and 597 2 380 pmole 32P incorporated per milligram of protein, respectively]. The mean ages of the “responder” subjects (4 men and 1 woman) and the “nonresponder” subjects (4 men and 2 women) also did not differ [27 + 6 (SE) and 28.8 2 7.5 years, respectively]. Molecular

Weights of Phosphorylated

Proteins

Autophosphorylation followed by SDS-PAGE and autoradiography reveals that angiotensin II increased the tyrosyl-phosphorylation of three proteins with molecular weights of 57 to 63 kDa (Fig. 2). DISCUSSION Many studies suggest that angiotensin converting enzyme and angiotensin II play a role in the inflammatory process. Activity of angiotensin converting en-

612 109 581 850

(3) (3) (3) (3)

AH

n.d. r 32 r 23 2 6 zt 22

M

-40 -29 296 -33

% Increase
P 1244 538 146 524 362

? + k 2 ?

31 41 20 217 I5

No AI1 (3) (2) (3) (3) (3)

M

2362 ‘704211 469 f 3178 k 642 -e

IO-’ 150 (3) (3) 62 (3) 623 (3) 18 (3)

AI1

90 31 221 506 77

% Increase

Stimulated with LPS

TABLE 1 Cell Tyrosine Kinase Activity in Healthy Subjects

<0.05 <0.025
P

Note. Cells were exposed to IO-” M angiotensin II (AH) and then stimulated with LPS (IO pg/ml) or not stimulated. Tyrosine kinase activity is expressed as mean 2 SE pmole “P incorporated/mg protein in replicate assays; n is in parentheses. P = probability by independent t test, no AI1 vs 10m5M AII. All subjects were healthy and on no medications. n.d. = no data, n.s. = not significant.

(3) (3) (2) (3)

nd. k 29 c 19 f 101 ” 38

1014 153 196 1274

2 3 4 5

I

lo-’

No AI1

Subject

of Adherent Mononuclear

Unstimulated

Augmentation

52

SIMON ET AL. TABLE 2 Adherent Mononuclear Cell Tyrosine Kinase Activity in “Nonresponder” Subjects

Lack of Augmentation Subject 6 7 8 9 10 11

AI1

No AI1 2494 147 154 326 229 232

f t ? + 2 k

70 17 12 10 131 52

(3) (3) (3) (3) (2) (3)

2435 164 158 332 106 182

+f ? 2 2 f

% Increase

129 28 24 I1 72 51

(3) (3) (3) (3) (3) (3)

-2 12 3 2 -54 -22

P

ns IIS

ns ns ns ns

Note. Cells were exposed to IO-’ M angiotensin II (AU) and then stimulated with LPS (10 &ml). Tyrosine kinase activity is expressed as mean * SE pmole “P incorporated/mg protein in replicate assays; n is in parentheses. P = probability by independent t test, no AI1 vs IO-’ M AII. All subjects were healthy and on no medications.

zyme is increased in the serum of patients with sarcoidosis (1) and leprosy (17). It is present in epithelioid cells of the sarcoid granuloma (2), in pulmonary alveolar macorphages (4,5), in Gaucher’s cells (18), and in blood monocytes from patients with sarcoidosis (3). Angiotensin II, a product of the converting enzyme, affects the function of many inflammatory cells. For example, it inhibits murine macrophage migration (8) and Fc receptor-mediated rosette formation (19), and modulates phagocytosis

900C 2 600e 0 0700E G P 600. s 'j Psooiz h 4002 0 300E 0

LOG

MOLAR

ANGIOTENSIN

II

FIG. 1. Dependence of the augmentation of monocyte tyrosine kinase activity upon the concentration of angiotensin II in two healthy subjects. Monocytes were exposed to angiotensin II and then stimulated with LPS (10 pg/ml). Tyrosine kinase activity is expressed as mean f SE pmole “P/mg protein. All assays were performed in triplicate. Neither subject was receiving any medication.

AI1 AUGMENTATION

FIG. 2. Adherent mononuclear cell Autoradiogram of “P-labeled proteins II (AH) or IO-’ M AI1 and stimulated phosphorylation of three proteins with

OF TYROSINE

KINASE

53

tyrosine kinase autophosphorylation followed by SDS-PAGE. from monocytes from patient 1 treated with no angiotensin with 10 pg/ml LPS. Angiotensin II increased the tyrosylmolecular weights of 57 to 63 kDa.

(9,lO). It also enhances cyclic AMP concentrations (20) and stimulates actin polymerization in granuloma macrophages (10). Tyrosine kinase catalyzes tyrosine phosphorylation of cellular proteins. Phosphorylation reactions may be an important mechanism in the regulation of the actions of many hormones (21). We therefore postulated that angiotensin II may act by altering adherent mononuclear cell tyrosine kinase activity. Cells from healthy subjects manifested two patterns. The cells of one group responded to angiotensin II with significant increases in tyrosine kinase activity, whereas those from the other group did not. This suggests that there may be “responder” and “nonresponder” subjects. Both groups were similar in age and sex. This phenomenon in the “responder” group was further studied. In the two subjects so studied, the increase in tyrosine kinase activity was dependent upon the concentration of angiotensin II. The tyrosine kinase activity of both subjects demonstrated peaks at lo-‘*, lo-‘, and 10e4 M angiotensin II. This suggests the possible presence of two or three different tyrosine kinases that are stimulated by angiotensin II. In addition, polyacrylamide gel electrophoresis revealed that the tyrosyl-phosphorylation of three cellular proteins with molecular weights of 57, 62, and 63 kDa was stimulated by angiotensin II. A monocyte phosphotyrosine protein with tyrosine kinase activity of similar molecular weight has been previously reported (22). T cells contain a 55 to 60kDa tyrosine kinase complexed to the CD4 molecule (23). Monocytes also possess CD4 (24,25). Whether monocyte CD4 is complexed to a tyrosine kinase remains to be determined.

54

SIMON ET AL.

Several studies suggest that alveolar macrophages from smokers are in an activated state (26-29). Alveolar macrophages from cigarette smokers possess increased angiotensin converting enzyme activity as compared with those from nonsmokers (5). This suggests the possibility that activated macrophages produce more angiotensin converting enzyme and angiotensin II. Angiotensin II production by granuloma macrophages has been demonstrated (30). Therefore angiotensin II production may be part of an autocrine amplification mechanism since angiotensin II augments monocyte tyrosine kinase actitity. This mechanism may be relevant only in active inflammation since the adherent mononuclear cells require activation with LPS before angiotensin II augmentation of tyrosine kinase activity occurs. SUMMARY The relationship of angiotensin converting enzyme activity and angiotensin II to the inflammatory process in diseases such as sarcoidosis remains unclear. We hypothesize that granuloma macrophages regulate inflammation by release of angiotensin converting enzyme, which produces angiotensin II, and that angiotensin II in turn modulates monocyte/macrophage activity. Since tyrosine kinase catalyzes phosphorylation of tyrosine residues in proteins and is important in signal transduction and cellular activation, we further postulated that monocyte tyrosine kinases may play a role in the regulation of this process. Mononuclear cells from 11 healthy subjects were assayed for tyrosine kinase activity in the presence and absence of angiotensin II. In addition, tyrosine-specific phosphorylation of cellular proteins was also determined. Angiotensin II increased tyrosine kinase activity in a concentration-dependent manner. The maximal stimulation, which varied from 31 to 506%, was achieved following incubation of cells with lop4 M angiotensin II. Angiotensin II also increased the tyrosyl-phosphorylation of three proteins with molecular weights of 57, 62, and 63 kDa. We conclude that tyrosine kinase activity of adherent mononuclear cells and tyrosine phosphorylation of certain protein(s) may be involved in angiotensin II regulation of inflammatory processes. ACKNOWLEDGMENTS This work was supported by the Medical Research Service of the Department of Veterans Affairs. This work was presented in part at the Midwest Section Meeting of the American Federation for Clinical Research in Chicago on November 9, 1989.

REFERENCES 1. Lieberman, J., Amer. J. Med. 59, 365 (1975). 2. Silverstein, E., Pertschuk, L. P., and Friedland, J., Proc. Natl. Acad. Sci. USA 76,6646 (1979). 3. Okabe, T., Yamagata, K., Fujisawa, M., Watanabe, J., Takaku, F., Lanzillo, J. J., and Fanburg, B. L., J. Clin. Invest. 75, 911 (1985). 4. Hinman, L. M., Stevens, C., Matthay, R. A., and Gee, J. B., Science 205, 202 (1979). 5. Sugiyama, Y., Yotsumoto, H., Okabe, T., and Takaku, F., Respiration 53, 153 (1988). 6. Dezso, B., Nielsen, A. H., and Poulsen, K., J. Cell Sci. 91, 155 (1988). 7. Dezso, B., Jacobsen, J., and Paulsen, K., J. Hypertension 7, 5 (1989). 8. Weinstock, J. V., and Blum, A. M., J. Zmmunol. 131, 2529 (1983).

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OF TYROSINE

KINASE

55

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Foris, G., Dezso, B., Medgyesi, G. A., and Fust, G., Immunology 48, 529 (1983). Weinstock, J. V., and Kassab, J. T., 1. Immunol. 132, 2598 (1984). Heldin, C.-H., and Westermark, B., Cell 37, 9 (1984). Majumdar, A. P. N., Edgerton, E. A., and Arlow, F. L., Biochim. Biophys. Acra 965,97 (1988). Bradford, M. M., Anal. Biochem. 72, 248 (1976). Majumdar, A. P. N., and Arlow, F. L., Amer. J. Physiol. 257, G554 (1989). Cooper, J. A., and Hunter, T., Mol. Cell. Viol. 1, 165 (1981). Dangott, L. J., Puett, D., and Melner, M. H., Biochim. Biophys. Actu 886, 187 (1986). Lieberman, J., and Rea, T. H., Ann. Intern. Med. 87, 422 (1977). Silverstein, E., Pertschuk, L. P., and Friedland, J., Amer. J. Med. 69, 408 (1980). Dezso, B., and Foris, G., Immunology 42, 277 (1981). Weinstock, J. V., and Kassab, J. T., Cell. Immunol. 89, 46 (1984). Cohen, P., Eur. .I. Biochem. 151, 439 (1985). Gee, C. E., Griffin, J., Sastre, L., Miller, L. J., Springer, T. A., Piwnica-Worms, H., and Roberts, T. M., Proc. Natl. Acad. Sci. USA 83, 5131 (1986). 23. Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schlossman, S. F., Proc. Natl. Acad. Sci. USA 85, 5190 (1988). 24. Wood, G. S., Warner, N. L., and Warnke, R. A., .I. Immunol. 131, 212 (1983). 25. Muscicki, R. A., Amento, E. P., Krane, S. M., Kurnick, J. T., and Calvin. R. B., J. Immunol. 131, 743 (1983).

26. Harris, J. O., Swenson, E. W., and Johnson, J. E., III, J. C/in. Invest. 49, 2086 (1970). 27. Hoidal, J. R., Fox, R. B., LeMarbe, P. A., Perri, R., and Repine, J. E., Amer. Rev. Respir. Dis.

123, 85 (1981).

28. Hoidal, J. R., and Niewoehner, D. E., Amer. Rev. Respir. Dis. 128, 548 (1982). 29. Baughman, R. P., Corser. B. C., Strohofer, S., and Hendricks, D., J. Lab. Clin. Med. (1986).

30. Weinstock, J. V., and Blum, A. M., Cell. Immunol.

89, 39 (1984).

107, 233