Transmembrane Signaling Changes with Aging” T. FULOP, JR.,”.‘ G. BARABAS,d ZS. VARGA,” J. CSONGOR,‘ M. HAUCK,” S. SZUCS,” I. SERES,” A. MOHACSI,” D. KEKESSY: J. P. DESPONT; L. ROBERT,^ AND A. P E N Y I G E ~ ”First Department of Medicine, dlnstitute of Biology, and ‘Central Research Laboratory University Medical School of Debrecen 4012 Debrecen POB 18, Hungary flnstitut Biome‘dical I227 Geneva, Switzerland gLaboratoire Biochimique du Tissu Conjunctif Universite‘ de Paris XII Paris, France
INTRODUCTION It is well known that we attribute to aging an alteration of the immune re~ p o n s e . ’ -Many ~ studies have been carried out to try to explain the age-related decline. Recently, it has been demonstrated that there are several intracellular biochemical signaling pathways linking receptors to effector function^.^,^ Few studies have been performed concerning the relationship of these fundamental cell functions to aging.6-8 In our previous studies using phagocytic cells and lymphocytes, we have demonstrated an alteration of transmembrane signaling in correlation with aging.9-” Recently, this observation was supported by It has been suggested that these alterations are due to changes in the amounts and possibly in the pattern and composition of G proteins, which transduce both activatory and inhibitory impulses from receptors on the cell surface to intracellular second-messenger systems. The a subunit of Gi can be ADP-ribosylated by using the toxin of Bordetella pertussis (PT). Similar ADP-ribosylation of the a subunit of G, is catalyzed by the toxin from Vibrio cholerae (CT). These toxins have been widely used for the identification of G,, and G,,, though it has become clear that they are not specific for them. The aim of the present study was to characterize the G proteins of human neutrophils and lymphocytes and to find out whether their amounts are altered in these cells from healthy young and elderly subjects.
“ This work was supported by grants from the Sandoz Foundation for Gerontological Research (SF 174) and the National Research Foundation (OTKA 1459). ‘To whom correspondence should be addressed. 165
166
ANNALS NEW YORK ACADEMY OF SCIENCES
MATERIALS AND METHODS Patients. Neutrophil granulocytes (PMNLs) and lymphocytes were obtained from 10 healthy young (aged 18-24 years) and elderly (aged 65-82) subjects after informed consent. The selection of persons was based on the criteria of good physical and mental states confirmed by careful clinical and laboratory investigation. Neutrophils were separated by Fycoll-Hypaque density centrifugation, followed by dextran sedimentation of the PMNL-rich pellet.9 Judged by morphological criteria, the neutrophil suspension was 95% pure, and 97% of the cells were viable as determined by Trypan blue exclusion. Lymphocytes were separated as described earlier. Measurement of inositol phosphate mobilization was carried out as published previously by us. lo ''C-FMLP Binding Assay. PMNLs (5 x lo6cells/ml) obtained from young and elderly people were incubated with increasing concentrations (0.1-50 nM) of I4CFMLP (specific activity 15.4 mcilmmol), for 60 min at 22°C. After incubation the cell suspension was centrifuged (1400 rpm, 10 min, 22°C) and washed twice with cold buffer. The cells were lysed with 0.3 M NaOH, filled with scintillation liquid, and transferred into an appropriate cuvette; and the activity of the samples was measured in a liquid scintillation counter. The unspecific binding of I4C-FMLP was determined in a parallel experiment in the presence of a 1000-fold excess of unlabeled FMLP. All experiments were performed in triplicate. The binding affinity of I4C-FMLP was calculated by Scatchard analysis. ADP-Ribosylation Assay. ADP-ribosylation of Gi, proteins by PT was carried out at 37°C for 60 min in a solution containing 20 mM Tris HCI (pH 7.3), 1 mM EDTA, 5 mM MgCI,, 0.1 mM CaCI,, 5 mM dithiothreitol, 20 mM thymidine, 1 mM ATP, 0.1 mM GTP, 3 pCi 32P-NAD(specific activity 36 mCi/mmol), and 0.1 pg activated PT (prepared by incubating the toxin with 20 mM dithiotreitol at 37°C for 30 min). The ADP ribosylation reaction was initiated by addition of the crude extracts of purified human lymphocytes and neutrophils in a total volume of 150 pl. The reaction was terminated by the addition of 150 pl ice-cold 20% (w/v) TCA, and the samples were kept on ice for 30 min. The precipitated proteins were centrifuged for 5 min at 4°C at 12000 g , then the pellet was washed twice with diethyl ether to remove the residual TCA. Finally, the samples were resuspended in 50 pl of 10 mM potassium phosphate buffer (pH 7.2) containing 1% (v/v) sarcosyl and 10% (v/v) a-mercaptoethanol; and after vigorous mixing this solution was supplemented with 50 pl of electrophoresis buffer (4% w/v SDS, 0.4 mM dithiothreitol, 40% w/v glycerol, and 0.004% w/v Coomassie Brilliant Blue in 20 mM potassium phosphate buffer, pH 7.2). The electrophoresis was performed as described by Laemmli.14 The gel was stained with Coomassie Brilliant Blue, dried under vacuum, and exposed to X-ray film for 3 days at -8O"C, using intensifier screens. The ADP ribosylation of G,, by CT was carried out according to the method of Gill et al.ls
RESULTS As has been published earlier, during FMLP, GTPaS, CD3, and elastin peptide stimulation, an age-dependent decrease in formation of second messengers has been demonstrated in PMNLs and lymphocytes with regard to both inositol phos-
FULOP et al.: TRANSMEMBRANE SIGNALING
167
phate formation and calcium metabolism. lo,ll It has also been demonstrated that the receptor affinity or number of receptors for IL-2, anti-CD3, and soluble elastin peptides was not altered with In the first series of our experiments we studied the effects of FMLP on PMNLs inositol trisphosphate (IP,) formation. It was found that elderly subjects may be divided into two groups: (1) “nonreactive,” in which practically no IP, formation under stimulation could be obtained; and (2) “reactive,” in which IP, formation was found, which was, however, significantly less than in PMNLs of young subjects (FIG.1). On the basis of these and previous results, we wondered whether any differences could exist between young and elderly subjects, as well as between the two groups of elderly, in receptor affinity or the number of FMLP receptors. Measuring the binding of I4C-FMLP to PMNLs of elderly “reactive” and “nonreactive” subjects, as well as to PMNLs of young subjects, we found that in receptor binding
a. young
b. old
FIGURE 1. Effect of lo-* M FMLP on inositol trisphosphate formation after IS s of stimulation in PMNLs of young (a) and elderly (b) subjects. The elderly group was divided into “reactive” (hatched colitmn) and “nonreactive” (striped column) subjects according to their capacity to produce IP, under stimulation.
affinity no measurable differences exist in either the low- or high-affinity FMLP receptors (FIG.2). These results strongly suggest that the alterations found in second messenger generation during aging are not due to the decreased binding of agonists to their receptors. The traditional means of identifying the G proteins is based on the ability of CT to catalyze the transfer of 32P-ADP-ribosefrom j*P-NAD+ to G,, and of PT to catalyze analogous ADP-ribosylation of Gi, and G,. The autoradiogram of ’*P-ADP-ribosylated proteins by CT from the crude extracts of human lymphocytes and PMNLs obtained from young and elderly individuals is shown in FIG.3. These polypeptides, specifically labeled in response to the presence of CT were a major polypeptide of 40 kDa and two much less prevalent components of 52 and 45 kDa, respectively, in lymphocytes of young subjects. In contrast, in lymphocytes of elderly subjects the major polypeptide
ANNALS NEW YORK ACADEMY OF SCIENCES
168
I
L
KA- 8370 nM
FIGURE 2. Binding of I4C-FMLPto polymorphonuclear leukocytes of young @/led circles) and elderly “reactive” (open circles) and “nonreactive” (triangles) subjects. Cells were incubated with increasing concentrations of I4C-FMLP in the presence and absence of unlabeled FMLP; the amount of specifically bound FMLP was calculated from these data. Each experiment was done in triplicate. Scatchard analysis of the binding data is shown in the inset.
was the 45-kDa polypeptide, and the two others were very weakly labeled. Autoradiograms of the gels showed clear differences in the patterns of the labeled bands depending on cell types and age. In PMNLs CT labeled quite strongly the 45-kDa band, mainly in elderly people; and the two others, 52 kDa and 40 kDa, were very weakly labeled, mainly in young subjects. These proteins may be three forms of
kDa
-66
-45
-”
-28
FIGURE 3. A typical autoradiogram of j2PADP-ribosylated proteins by activated cholera toxin (CT) from the crude extract of human lymphocytes and granulocytes obtained from young and elderly individuals. The j2PADP-ribosylated proteins were separated by SDS-PAGE and analyzed by autoradiography. Lane 1: CT-treated crude extract of lymphocytes from young individuals; lane 2: CT-treated crude extract of lymphocytes from elderly individuals; lane J: CT-treated crude extract of granulocytes from young individuals; lane 4: CT-treated crude extract of granulocytes from elderly individuals. The position of molecular mass markers are indicated on the right side of the figure.
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Gsa, The autoradiogram of 32P-ADP-ribosylated proteins by PT from the crude extracts of human lymphocytes and PMNLs obtained from young and elderly people is shown in FIG.4. No age-related pattern of changes could be demonstrated, while differences could be observed among the types of cells. In lymphocytes of both age groups the major F'T-labeled peptide was 41 kDa; however, the label intensity increased with aging, probably corresponding to Gi,. In PMNLs the major PT-labeled polypeptide was 40 kDa, probably corresponding to Goa.
1.
8. kDa
-
- 66 - 45
-4 0 -29 -21
-
-14
FIGURE 4. A typical autoradiogram of '!P-ADP-ribosylated proteins by activated pertussis toxin (PT) from the crude extract of human lymphocytes and granulocytes obtained from young and elderly individuals. The 3ZP-ADP-ribosylatedproteins were separated by SDSPAGE and analyzed by autoradiography. Lanes 1 and 8: The position of molecular mass markers; lane 2: PT-treated crude extract of lymphocytes from young individuals; lane 3: PT-treated crude extract of lymphocytes from elderly individuals; lanes 4 and 6: PT-treated crude extract of granulocytes from young individuals (80-pg and 150-pg protein/sample, respectively); lanes 5 and 7: PT-treated crude extract of granulocytes from elderly individuals (SO-pg and 150-pg protein/sample, respectively).
DISCUSSION We have demonstrated altered immune functions with It is not yet known what the exact underlying mechanism of these changes is. The decrease in the number of receptors was thought to be involved.ln In the present study we have once again demonstrated that no decrease in FMLP receptor affinity and number can be observed on PMNLs with aging. This is the case even when IP3 formation is altered to varied extents with aging. Previously, we have demonstrated in PMNLs and lymphocytes that transmem-
170
ANNALS NEW YORK ACADEMY OF SCIENCES
brane signaling is altered with aging, and as a consequence second messenger formation is ~hanged'*"*'~-that is, IP, formation is decreased, and [Ca2+Iimetabolism and cyclic nucleotide formation are altered. The PKC translocation is also changed with aging, more markedly in PMNLs than in lymphocytes (unpublished data). Taking into account all the above-mentioned data, we thought that one possible alteration that could underlie the changed transmembrane signaling with aging is the change in G protein pattern or quantity. G proteins transduce signals from surface receptors to intracellular effectors such as adenylate cyclase and phospholipase C. G proteins are also involved in regulation of ion channels and exocytosis, cell growth, T cell maturation, and various p a t h o l o g i e ~ . ' ~ . ~ ~ We attribute to aging an increase of CT- and PT-ADP ribosylated G proteins in PMNLs (unpublished data). An alternative method for identifying GTP binding proteins, involved in receptor-activated signal transduction, makes use of bacterial toxins, such as CT, PT, and BT. In this study, we determined that CT catalyzes the ADP-ribosylation of three distinct proteins (FIG. 3), having molecular masses of 52,45, and 40 kDa, and that their distribution depends on cell type and age of subject. In lymphocytes of young individuals the most prominent band is a protein of 40 kDa, corresponding most probably to a Gi, protein. In lymphocytes of elderly subjects the most abundant is the 45-kDa protein, corresponding most probably to Gsa;the others are barely present. In PMNLs of young as well as of elderly subjects the most abundant is the 45-kDa protein. While in PMNLs of young subjects we observed only the 45-kDa band, in PMNLs of elderly subjects all three bands could be detected. FT catalyzes the ADP ribosylation of the Gi, subunit and thereby modifies its activity. In particular, PT causes the ADP ribosylation of a 41-kDa subunit of Gia. The PT-mediated inactivation of G, leads to the inhibition of a variety of cellular activities.10s2'In this study we have determined that PT leads to ADP ribosylation of a 41-kDa protein in lymphocytes and a 40-kDa protein in PMNLs of young and elderly subjects. This 41-kDa protein corresponds most probably to aGi2. The 40kDa protein in PMNLs was demonstrated by Dickey er a/.zz In summary, the present data indicate changes in quantity and patterns of G proteins in membranes of lymphocytes and PMNLs of elderly subjects. These changes are not uniform in these two types of cells. Nevertheless, the observed changes could at least to some extent explain different degrees of responsiveness of lymphocytes and PMNLs to various specific stimuli with aging. The exact identification of G subunit changes with aging is under current investigation in our laboratory.
SUMMARY Altered immune response and transmembrane signaling with aging has previously been demonstrated. The aim of the present study was to characterize PMNLs and lymphocyte G proteins and to determine whether their relative amounts are altered with aging. First we studied the effects of FMLP on PMNLs IP3 formation. It was found that in any group of elderly the PMNLs IP, formation was significantly decreased compared to that of young subjects. In FMLP receptor binding affinity no measurable difference exists in either low- or high-affinity FMLP receptors. The autoradiogram of 32P-ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40 kDa, and two
FULOP et a/.: TRANSMEMBRANE SIGNALING
171
much less prevalent components of 52 and 45 kDa. In contrast, in lymphocytes of elderly subjects the major polypeptide was 45 kDa, and the two others were very weakly labeled. In PMNLs, CT labeled the 45-kDa band quite strongly, mainly in the elderly, and the 52- and 40-kDa bands were very weakly labeled, mainly in young subjects. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. ACKNOWLEDGMENTS We are particularly grateful for the technical assistance of Mrs. Magda Nagy, Mrs. Marika Kovacs, and Mrs. Gyongyi Sallai. REFERENCES I.
2. 3. 4. 5.
6. 7. 8. 9. 10.
II. 12. 13. 14.
IS. 16. 17. 18.
19. 20. 21. 22.
T. TANAKA, K. YOSHIZAKI, N. NISHINEGORO,S., H. HARA,S . MIYATA,0. SAIKI, MOTO & S. KISHIMOTO. 1987. Mech. Ageing Dev. 3 9 263-274. LIGHTHART, G. J., P. C. VANVLOKHOREN, H. R. E. SCHUIT& W. HUMANS.1986. Immunology 59: 353-367. FULOP, T., JR., G. FORIS,I. WORUM& A. LEOVEY.1984. Int. Arch. Allergy Appl. Immunol. 7 4 76-79. UERRIDGE, M. J . 1987. Annu. Rev. Biochem. 56: 159-193. FREISSMUTH, M.. P. J. CASEY& A. G. GILMAN.1989. FASEB J. 3: 2125-2131. LIPSCHITZ. D. A., K. B. UDUPA& L. A. BOXER.1988. Blood 71: 659-665. S. A. HARRISON, M. A. BUCHHOLZ & A. A. NORDIN. PROUST,J. J., C. R. FILBURN, 1987. J. Immunol. 139: 1472-1478. ROTH,G. S. 1986. Fed. Proc. 45: 60-64. FULOP, T., JR., G. FORIS,1. WORUM& A. LEOVEY.1985. Clin. Exp. Immunol. 61: 425-432. FULOP. T., JR., Zs. VARGA,J. CSONGOR,G. FORIS& A. LEOVEY.1989. FEBS Lett. 245: 249-252. A. M. ZAIA, L. BENE,T. FULOP,JR., A. LEOVEY,N. VARGA,Zs., N. BRESSANI, FABRIS & S. DAMJANOVICH. 1990. Immunol. Lett. U:275-280. THOMAN. M. L. & W. 0. WEIGLE.1989. I n Advances in Immunology, Vol. 46: D. Dixon. Ed.: 221-259. Academic Press. New York, NY. NORDIN, A. A. & J. J. PROUST.1987. Endocrinol. Metab. Clin. North Am. 16 919-945. U . K. 1970. Nature 227: 680-685. LAEMMLI, GILL,D. M. & M. WOOLKALIS. 1988. Methods Enzymol. 165: 235-245. FULOP,T., JR., M. UTSUYAMA, & K. HIROKAWA. 1991. J. Clin. Lab. Immunol. 34: 3 1-36. VARGA,Zs., M. P. JACOB,J. CSONGOR, L. ROBERT,A. LEOVEY & T. FULOP, JR. 1990. Mech. Ageing Dev. 52: 61-70. 0. SAIKI, T. TANAKA, K. YOSHIZAKI,T. IGARASHI NEGORO,S., H. HARA,S. MIYATA, & S. KISHIMOTO. 1986. Mech. Ageing Dev. 36: 223-241. PESSA-MORIKAWA, T., T. MUSTELIN& L. C. ANDERSON.1990. J. Immunol. 144: 2690-2695. MILLIGAN, G. & E. D. SAGGERSON. 1990. Biochem. J. 270 765-769. S. J., R. W. POUGHERTY, E. G. LAPETINA & J. E. NIEDEL.1985. Proc. Natl. BRANDT, Acad. Sci. USA 82: 3277-3280. , C. WILLIAMSON & J. NAVARRO. 1987. FEBS Lett. 219: DICKEY, F. B., H. J. P Y U NK. 289-292.
INTRODUCTION It is well known that we attribute to aging an alteration of the immune re~ p o n s e . ’ -Many ~ studies have been carried out to try to explain the age-related decline. Recently, it has been demonstrated that there are several intracellular biochemical signaling pathways linking receptors to effector function^.^,^ Few studies have been performed concerning the relationship of these fundamental cell functions to aging.6-8 In our previous studies using phagocytic cells and lymphocytes, we have demonstrated an alteration of transmembrane signaling in correlation with aging.9-” Recently, this observation was supported by It has been suggested that these alterations are due to changes in the amounts and possibly in the pattern and composition of G proteins, which transduce both activatory and inhibitory impulses from receptors on the cell surface to intracellular second-messenger systems. The a subunit of Gi can be ADP-ribosylated by using the toxin of Bordetella pertussis (PT). Similar ADP-ribosylation of the a subunit of G, is catalyzed by the toxin from Vibrio cholerae (CT). These toxins have been widely used for the identification of G,, and G,,, though it has become clear that they are not specific for them. The aim of the present study was to characterize the G proteins of human neutrophils and lymphocytes and to find out whether their amounts are altered in these cells from healthy young and elderly subjects.
“ This work was supported by grants from the Sandoz Foundation for Gerontological Research (SF 174) and the National Research Foundation (OTKA 1459). ‘To whom correspondence should be addressed. 165
166
ANNALS NEW YORK ACADEMY OF SCIENCES
MATERIALS AND METHODS Patients. Neutrophil granulocytes (PMNLs) and lymphocytes were obtained from 10 healthy young (aged 18-24 years) and elderly (aged 65-82) subjects after informed consent. The selection of persons was based on the criteria of good physical and mental states confirmed by careful clinical and laboratory investigation. Neutrophils were separated by Fycoll-Hypaque density centrifugation, followed by dextran sedimentation of the PMNL-rich pellet.9 Judged by morphological criteria, the neutrophil suspension was 95% pure, and 97% of the cells were viable as determined by Trypan blue exclusion. Lymphocytes were separated as described earlier. Measurement of inositol phosphate mobilization was carried out as published previously by us. lo ''C-FMLP Binding Assay. PMNLs (5 x lo6cells/ml) obtained from young and elderly people were incubated with increasing concentrations (0.1-50 nM) of I4CFMLP (specific activity 15.4 mcilmmol), for 60 min at 22°C. After incubation the cell suspension was centrifuged (1400 rpm, 10 min, 22°C) and washed twice with cold buffer. The cells were lysed with 0.3 M NaOH, filled with scintillation liquid, and transferred into an appropriate cuvette; and the activity of the samples was measured in a liquid scintillation counter. The unspecific binding of I4C-FMLP was determined in a parallel experiment in the presence of a 1000-fold excess of unlabeled FMLP. All experiments were performed in triplicate. The binding affinity of I4C-FMLP was calculated by Scatchard analysis. ADP-Ribosylation Assay. ADP-ribosylation of Gi, proteins by PT was carried out at 37°C for 60 min in a solution containing 20 mM Tris HCI (pH 7.3), 1 mM EDTA, 5 mM MgCI,, 0.1 mM CaCI,, 5 mM dithiothreitol, 20 mM thymidine, 1 mM ATP, 0.1 mM GTP, 3 pCi 32P-NAD(specific activity 36 mCi/mmol), and 0.1 pg activated PT (prepared by incubating the toxin with 20 mM dithiotreitol at 37°C for 30 min). The ADP ribosylation reaction was initiated by addition of the crude extracts of purified human lymphocytes and neutrophils in a total volume of 150 pl. The reaction was terminated by the addition of 150 pl ice-cold 20% (w/v) TCA, and the samples were kept on ice for 30 min. The precipitated proteins were centrifuged for 5 min at 4°C at 12000 g , then the pellet was washed twice with diethyl ether to remove the residual TCA. Finally, the samples were resuspended in 50 pl of 10 mM potassium phosphate buffer (pH 7.2) containing 1% (v/v) sarcosyl and 10% (v/v) a-mercaptoethanol; and after vigorous mixing this solution was supplemented with 50 pl of electrophoresis buffer (4% w/v SDS, 0.4 mM dithiothreitol, 40% w/v glycerol, and 0.004% w/v Coomassie Brilliant Blue in 20 mM potassium phosphate buffer, pH 7.2). The electrophoresis was performed as described by Laemmli.14 The gel was stained with Coomassie Brilliant Blue, dried under vacuum, and exposed to X-ray film for 3 days at -8O"C, using intensifier screens. The ADP ribosylation of G,, by CT was carried out according to the method of Gill et al.ls
RESULTS As has been published earlier, during FMLP, GTPaS, CD3, and elastin peptide stimulation, an age-dependent decrease in formation of second messengers has been demonstrated in PMNLs and lymphocytes with regard to both inositol phos-
FULOP et al.: TRANSMEMBRANE SIGNALING
167
phate formation and calcium metabolism. lo,ll It has also been demonstrated that the receptor affinity or number of receptors for IL-2, anti-CD3, and soluble elastin peptides was not altered with In the first series of our experiments we studied the effects of FMLP on PMNLs inositol trisphosphate (IP,) formation. It was found that elderly subjects may be divided into two groups: (1) “nonreactive,” in which practically no IP, formation under stimulation could be obtained; and (2) “reactive,” in which IP, formation was found, which was, however, significantly less than in PMNLs of young subjects (FIG.1). On the basis of these and previous results, we wondered whether any differences could exist between young and elderly subjects, as well as between the two groups of elderly, in receptor affinity or the number of FMLP receptors. Measuring the binding of I4C-FMLP to PMNLs of elderly “reactive” and “nonreactive” subjects, as well as to PMNLs of young subjects, we found that in receptor binding
a. young
b. old
FIGURE 1. Effect of lo-* M FMLP on inositol trisphosphate formation after IS s of stimulation in PMNLs of young (a) and elderly (b) subjects. The elderly group was divided into “reactive” (hatched colitmn) and “nonreactive” (striped column) subjects according to their capacity to produce IP, under stimulation.
affinity no measurable differences exist in either the low- or high-affinity FMLP receptors (FIG.2). These results strongly suggest that the alterations found in second messenger generation during aging are not due to the decreased binding of agonists to their receptors. The traditional means of identifying the G proteins is based on the ability of CT to catalyze the transfer of 32P-ADP-ribosefrom j*P-NAD+ to G,, and of PT to catalyze analogous ADP-ribosylation of Gi, and G,. The autoradiogram of ’*P-ADP-ribosylated proteins by CT from the crude extracts of human lymphocytes and PMNLs obtained from young and elderly individuals is shown in FIG.3. These polypeptides, specifically labeled in response to the presence of CT were a major polypeptide of 40 kDa and two much less prevalent components of 52 and 45 kDa, respectively, in lymphocytes of young subjects. In contrast, in lymphocytes of elderly subjects the major polypeptide
ANNALS NEW YORK ACADEMY OF SCIENCES
168
I
L
KA- 8370 nM
FIGURE 2. Binding of I4C-FMLPto polymorphonuclear leukocytes of young @/led circles) and elderly “reactive” (open circles) and “nonreactive” (triangles) subjects. Cells were incubated with increasing concentrations of I4C-FMLP in the presence and absence of unlabeled FMLP; the amount of specifically bound FMLP was calculated from these data. Each experiment was done in triplicate. Scatchard analysis of the binding data is shown in the inset.
was the 45-kDa polypeptide, and the two others were very weakly labeled. Autoradiograms of the gels showed clear differences in the patterns of the labeled bands depending on cell types and age. In PMNLs CT labeled quite strongly the 45-kDa band, mainly in elderly people; and the two others, 52 kDa and 40 kDa, were very weakly labeled, mainly in young subjects. These proteins may be three forms of
kDa
-66
-45
-”
-28
FIGURE 3. A typical autoradiogram of j2PADP-ribosylated proteins by activated cholera toxin (CT) from the crude extract of human lymphocytes and granulocytes obtained from young and elderly individuals. The j2PADP-ribosylated proteins were separated by SDS-PAGE and analyzed by autoradiography. Lane 1: CT-treated crude extract of lymphocytes from young individuals; lane 2: CT-treated crude extract of lymphocytes from elderly individuals; lane J: CT-treated crude extract of granulocytes from young individuals; lane 4: CT-treated crude extract of granulocytes from elderly individuals. The position of molecular mass markers are indicated on the right side of the figure.
FULOP er al.: TRANSMEMBRANE SIGNALING
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Gsa, The autoradiogram of 32P-ADP-ribosylated proteins by PT from the crude extracts of human lymphocytes and PMNLs obtained from young and elderly people is shown in FIG.4. No age-related pattern of changes could be demonstrated, while differences could be observed among the types of cells. In lymphocytes of both age groups the major F'T-labeled peptide was 41 kDa; however, the label intensity increased with aging, probably corresponding to Gi,. In PMNLs the major PT-labeled polypeptide was 40 kDa, probably corresponding to Goa.
1.
8. kDa
-
- 66 - 45
-4 0 -29 -21
-
-14
FIGURE 4. A typical autoradiogram of '!P-ADP-ribosylated proteins by activated pertussis toxin (PT) from the crude extract of human lymphocytes and granulocytes obtained from young and elderly individuals. The 3ZP-ADP-ribosylatedproteins were separated by SDSPAGE and analyzed by autoradiography. Lanes 1 and 8: The position of molecular mass markers; lane 2: PT-treated crude extract of lymphocytes from young individuals; lane 3: PT-treated crude extract of lymphocytes from elderly individuals; lanes 4 and 6: PT-treated crude extract of granulocytes from young individuals (80-pg and 150-pg protein/sample, respectively); lanes 5 and 7: PT-treated crude extract of granulocytes from elderly individuals (SO-pg and 150-pg protein/sample, respectively).
DISCUSSION We have demonstrated altered immune functions with It is not yet known what the exact underlying mechanism of these changes is. The decrease in the number of receptors was thought to be involved.ln In the present study we have once again demonstrated that no decrease in FMLP receptor affinity and number can be observed on PMNLs with aging. This is the case even when IP3 formation is altered to varied extents with aging. Previously, we have demonstrated in PMNLs and lymphocytes that transmem-
170
ANNALS NEW YORK ACADEMY OF SCIENCES
brane signaling is altered with aging, and as a consequence second messenger formation is ~hanged'*"*'~-that is, IP, formation is decreased, and [Ca2+Iimetabolism and cyclic nucleotide formation are altered. The PKC translocation is also changed with aging, more markedly in PMNLs than in lymphocytes (unpublished data). Taking into account all the above-mentioned data, we thought that one possible alteration that could underlie the changed transmembrane signaling with aging is the change in G protein pattern or quantity. G proteins transduce signals from surface receptors to intracellular effectors such as adenylate cyclase and phospholipase C. G proteins are also involved in regulation of ion channels and exocytosis, cell growth, T cell maturation, and various p a t h o l o g i e ~ . ' ~ . ~ ~ We attribute to aging an increase of CT- and PT-ADP ribosylated G proteins in PMNLs (unpublished data). An alternative method for identifying GTP binding proteins, involved in receptor-activated signal transduction, makes use of bacterial toxins, such as CT, PT, and BT. In this study, we determined that CT catalyzes the ADP-ribosylation of three distinct proteins (FIG. 3), having molecular masses of 52,45, and 40 kDa, and that their distribution depends on cell type and age of subject. In lymphocytes of young individuals the most prominent band is a protein of 40 kDa, corresponding most probably to a Gi, protein. In lymphocytes of elderly subjects the most abundant is the 45-kDa protein, corresponding most probably to Gsa;the others are barely present. In PMNLs of young as well as of elderly subjects the most abundant is the 45-kDa protein. While in PMNLs of young subjects we observed only the 45-kDa band, in PMNLs of elderly subjects all three bands could be detected. FT catalyzes the ADP ribosylation of the Gi, subunit and thereby modifies its activity. In particular, PT causes the ADP ribosylation of a 41-kDa subunit of Gia. The PT-mediated inactivation of G, leads to the inhibition of a variety of cellular activities.10s2'In this study we have determined that PT leads to ADP ribosylation of a 41-kDa protein in lymphocytes and a 40-kDa protein in PMNLs of young and elderly subjects. This 41-kDa protein corresponds most probably to aGi2. The 40kDa protein in PMNLs was demonstrated by Dickey er a/.zz In summary, the present data indicate changes in quantity and patterns of G proteins in membranes of lymphocytes and PMNLs of elderly subjects. These changes are not uniform in these two types of cells. Nevertheless, the observed changes could at least to some extent explain different degrees of responsiveness of lymphocytes and PMNLs to various specific stimuli with aging. The exact identification of G subunit changes with aging is under current investigation in our laboratory.
SUMMARY Altered immune response and transmembrane signaling with aging has previously been demonstrated. The aim of the present study was to characterize PMNLs and lymphocyte G proteins and to determine whether their relative amounts are altered with aging. First we studied the effects of FMLP on PMNLs IP3 formation. It was found that in any group of elderly the PMNLs IP, formation was significantly decreased compared to that of young subjects. In FMLP receptor binding affinity no measurable difference exists in either low- or high-affinity FMLP receptors. The autoradiogram of 32P-ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40 kDa, and two
FULOP et a/.: TRANSMEMBRANE SIGNALING
171
much less prevalent components of 52 and 45 kDa. In contrast, in lymphocytes of elderly subjects the major polypeptide was 45 kDa, and the two others were very weakly labeled. In PMNLs, CT labeled the 45-kDa band quite strongly, mainly in the elderly, and the 52- and 40-kDa bands were very weakly labeled, mainly in young subjects. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. ACKNOWLEDGMENTS We are particularly grateful for the technical assistance of Mrs. Magda Nagy, Mrs. Marika Kovacs, and Mrs. Gyongyi Sallai. REFERENCES I.
2. 3. 4. 5.
6. 7. 8. 9. 10.
II. 12. 13. 14.
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