The Response of Human Lymphocytes to Phytohemagglutinin Is Impaired at Different Levels during Aging" CARL0 PIERI,h RINA RECCHIONI,h FAUSTO MORONI,b FIORELLA MARCHESELLI,b AND SANDOR DAMJANOVICH' hCytoIogy Center Department of Gerontological Research Italian National Research Centers on Aging (I.N.R.C.A.) 60121 Ancona, Italy CDepartmentof Biophysics University Medical School of Debrecen H-4012 Debrecen, Hungary
INTRODUCTION It has long been recognized that the responsiveness of lymphocytes to mitogenic stimulation is impaired in elderly human patients.'-6 Several hypotheses have been proposed to explain the mechanism of such an impairment. The increased microviscosity of the membrane lipid domain may be (at least partially) responsible for the age-dependent decrease of the lymphocyte re~ponse.~.' Support for this hypothesis stems from the observation that preculturing the lymphocytes from old donors for 48 hours in culture medium containing 10% human serum resulted in a decrease of membrane microviscosity and an increase of the response to PHA.8 Other authors pointed out that the impairment was at the level of early signal tran~duction,~ genomic activation,I0.I1or protein synthesis.I2 The response of lymphocytes to mitogenic or alloantigenic stimulation is very complex. More than 70 molecules are specifically regulated during the time kinetics of this p r o c e ~ s . In ' ~ principle, any impairment of such a regulatory step may have a causative relationship with the age-dependent decrease of the response observed in human lymphocytes. Although the age-related decrease of many parameters has been well documented (see for review Refs. 14, 15), few studies have examined the individual changes and the correlations between different parameters in each individual. In the present work, four different parameters have been compared that reflect different stages in the progression of lymphocyte activation-namely , the modification of cell membrane structure, the c-myc and c-myb expression, and the incorporation of labeled thymidine into newly synthesized DNA. In addition, data are presented on the responsiveness of the membrane potential of peripheral blood lymphocytes to depolarizing conditions as well as to PHA treatment.
a
This work was supported in part by a grant to C.P. from the National Research Council. 110
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MEMBRANE POTENTIAL STUDIES The binding of alloantigens or mitogenic lectin to the lymphocyte plasma membrane results in, among other things, changes of ion channel activities and of membrane potential. l6 The increase of intracellular free Ca+ induced by mitogens has already been extensively investigated: however, conflicting data have been reported on the age dependency of this parameter in h ~ m a n s . ~ . ~ 'As - ' *regards the alteration of membrane potential, a repolarizing effect of PHA treatment has been demonstrated,16 but data on the effect of aging on this parameter are lacking. Recently, we demonstrated the presence of ligand- and voltage-gated Na' channels in human lymphocytes using bretylium tosylate (BT), a quaternary ammoniumion. as ligand. BT treatment resulted in an increase of intracellular Na' content, with a consequent activation of Na'-K+-ATPase pump and repolan~ation.'~ This sensitive mechanism was found to be impaired by aging.?O In the present paper we report that the extent of PHA-induced repolarization of slightly depolarized lymphocytes depends on the age of the donors. To measure the membrane potential changes, the fluorescent probe oxonol [bis ( I ,3, dibutylbarbituric acid-(S)-trimethilene oxonol)] was applied, as described in previous papers.19-?()It has to be noted that the repolarization of lymphocytes with PHA is more complex than that induced by BT. Indeed, although complete depolarization inhibits the Ca' influx pathway,?' it was not able to influence mitogen-induced internal calcium release.?? Among conditions that partially depolarize cells, an increase of free intracellular Ca' concentration occurs (see in Ref. 22: we also have similar data not shown here) subsequent to mitogenic stimulation. Thus, at least two mechanisms may be responsible for the PHA-induced repolarization. According to our model, one of these mechanisms is the opening of voltageand ligand- (mitogen) gated Na+ channels and the activation thereby of Na+/K'dependent ATP-ase: the second mechanism is the opening of Ca'-activated K' channels, which also proved to be potential sensitive in lymphocytes.23 FIGURE 1 shows a typical example of the repolarizing effect of PHA in human lymphocytes from young (A) and old (B) persons. The increase or the decrease of fluorescence (channel number) is indicative of depolarization and repolarization, respectively. The bis-oxonol that provides the fluorescence signal is negatively charged: thus the fluorescence intensity and membrane potential of the cell have an inverse relationship. The shaded histograms represent the cells depolarized by increasing the extracellular K + from 5 to 73 mM, an isosmotic mechanism. Since the basal resting membrane potential (i.e., the fluorescence measured at 5 mM extracellular K') is not different in cells from young and old subjects (see Ref. 20; we have similar data not shown here), it is easy to conclude that the response of the old cells to the depolarizing conditions is lower than that of young ones. Although the membrane permeability could be an important factor in this phenomenon, a more complex mechanism cannot yet be excluded. Addition of PHA to the depolarized cells resulted in a repolarization (open histograms), the extent of which is again lower in old (B) as compared to young lymphocytes (A). TABLE I summarizes the results of the analysis of lymphocytes from 10 young and 10 old persons. The positive figures indicate a shift of mean fluorescence of the oxonol to the right (depolarization) and the negative figures the extent of the shift to the left (repolarization). Both the responses are impaired by aging, which supports the idea that they were initiated by an alteration of ion channel activities. Thus, aging may impair the response of lymphocytes to mitogens at a very early phase of the activation process. +
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Fluorescence in arbitrary units [channel No) FIGURE 1. Oxonol fluorescence histograms of lymphocytes from young (A) and old (B) subjects. Shaded and open histograms represent the cells depolarized by isosmotic increase of extracellular K + (73 mM) and subsequent incubation with PHA for five minutes at 37"C, respectively.
TABLE 1. Shift of Mean Oxonol Fluorescence in Young and Old Lymphocytes Youne K5-K73 K73-PHA
+48.5 -33.8
2 f
Old 1.4 2.8
+38.2 -20.5
If. f
3.0 2.6
p p
< 0.001 < 0.001
NOTE: Entries are mean If. SEM of 10 experiments. K5-K73 indicates the increase of mean oxonol fluorescence (depolarization) upon the isotonic increase of extracellular K+ concentration from 5 mM to 73 mM. K73-PHA indicates the decrease of mean oxonol fluorescence subsequent to PHA addition to the culture medium containing 73 mM K + as in FIG. I.
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ACTIVATION OF CELL MEMBRANE AND GENOMIC LEVELS As another early event, the binding of the stimulatory ligand to the lymphocytes’ plasma membrane leads to well-defined structural changes. We utilized these changes, occurring in the lipid packing, to monitor the percentage of lymphocytes activated by I-hour exposure to PHA at 37°C. For this purpose the uptake of merocyanine 540 (MC 5401, a fluorescent probe that preferentially binds to disordered membranes, was used.24-25 In addition, we determined the quantity of proteins, encoded by the activated c-myc and c-myb protooncogenes, which can also serve as markers of Go/G,and G,,/S probe transition, respectively. Taking these three measured parameters together, they provide information on the progression of cell activation processes for each individual cell. The results are presented in FIGURE 2. Since they were able to increase the amount of MC 540 bound to their membranes (i.e., the percentage of activated cells), the lymphocytes had a wider
OLD
YOUNG
1
O’
50 -
60
-ln
:
40:
e 0 &
c
30-
:
20-
I
a
=
. 10-
n,
FIGURE 2. Percent of the cells from young and old individuals activated by PHA stimulation as measured by MC 540 uptake and by c-myc- and c-myb-encoded protein expression.
distribution in old samples (range 22-54, mean 2 SEM 38.0 2 3.7) than in young samples (range 34-52, mean SEM 45.4 1.9). Although the difference between the means was not statistically significant, it is worthy of note that in 4 out of 10 old samples the percentage of activated lymphocytes was lower than the lowest percentage for the young samples. This suggests a delay of the response and/or an impairment of the mechanisms leading to the modification of membrane lipid packing for these samples. Examining a higher number of samples is likely to yield statistically significant differences. The presence of the c-myc-coded protein was assessed by indirect immunofluorescence, determined 16 hours after PHA stimulation. Rabbit polyclonal antibodies against a bacterially expressed protein encoded by exon, of the human c-myc gene (Medac, Molecular Biology, Germany) was used as first antibody. Fluoresceinated goat anti-rabbit IgG was the second antibody. In general, in the young samples the percentage of cells showing positivity for
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c-myc protein expression was higher than that measured with MC 540, suggesting that an increasing number of lymphocytes was activated by PHA later than one hour after exposure. On the contrary, in the lymphocytes from old donors, only 3 out of 10 samples showed a higher percentage of cells expressing c-myc than those measured by MC 540 incorporation. In the other samples, a more-or-less expressed decrease in the amount of positive cells was observed. It is interesting to note that in half of the samples from old individuals the percentage of cmyc-positive cells fell in the range that was characteristic for cells from the young. However, if one considers the whole population, the difference between the means was statistically significant (mean 2 SEM 52.2 f 2.2 in the young vs. 37.3 f 4.4 in the old, p < 0.01). The general meaning of these data is that the activation of the c-myc gene andlor the consequent synthesis of the specific protein have been impaired by aging; this is in agreement also with other authors.IoJ1However, this alteration is not an all-or-nothing phenomenon. As regards the number of cells expressing the c-myb-encoded protein, this parameter was determined 66 hours after PHA stimulation by indirect immunofluorescence, using as first antibody a polyclonal rabbit antibody to human c-myb protein (Medac, Molecular Biology, Germany). Fluoresceinated goat anti-rabbit IgG was the second antibody. As shown in FIGURE 2, not all the lymphocytes expressing c-myc protein were able to express also the c-myb-encoded one, and the decrease in the percentage of cells that lost this capability was higher in the old ( - 10.2%) than in young sample (-6.5%). Interestingly enough, a strong decrease in the number of cells positive for c-myb was observed from young to old populations (mean k SEM 45.7 f 2.5 and 27.1 f 3.2, in young and old lymphocytes, respectively; p < 0.001). A likely explanation for this decrease can be found at the level of regulation. It is well established, indeed, that c-myb gene expression is preceded by the binding of IL-2 to its receptor,26and both IL-2 synthesis and high-affinity IL-2 receptor expression seem to be impaired during aging (for review see Ref. 15).
'H-THYMIDINE INCORPORATION AND ITS CORRELATION WITH C-MYC AND C-MYB PROTEIN EXPRESSION An important question to be answered is whether the observed decrease in cmyc and c-myb expression could account for the decreased DNA synthesis observed in stimulated lymphocytes during aging. In order to answer this question we calculated the amount of 3H-thymidine incorporated by the cells expressing the c-myc- and c-myb-encoded proteins. The results are shown in TABLE2. The difference between the young and old populations observed for these parameters when the c-myc-positive cells were taken into account can easily been explained by the age-dependent decrease of a number of factors, such as IL-2 receptor expression, 11-2 synthesis (for review see Ref. 15), and c-myb activation (present paper), which follow c-myc activation and precede and are necessary for DNA synthesis. We observed that a difference still existed between young and old cells in the amount of labeled thymidine incorporated into the newly synthesized DNA even in the case of c-myb-positive cells. This finding suggested that despite the presence of the c-myb protein, not all of the cells from old donors are able to progress from G I to S phase, because of impairment(s) at level(s) different from those taken into account in the present paper. On the other hand, considering the lymphocytes from the old subject labeled with the asterisk in FIGURE 2, all the
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parameters analyzed fell in the ranges characteristic of the young population. Thus we could not explain the reduced 3H-thymidine uptake of this old sample as compared to the young one analyzed on the same day (125 x lo3cpm vs. 194 x lo3 cpm, respectively). A PARTICULAR CASE OF AGE-DEPENDENT ALTERATION OF
CELLULAR PARAMETERS Apart from the data presented above, another parameter has been analyzed in the lymphocytes of the same young and old subjects (mitochondrial activation and its results will be reported in detail elsewhere2'). This parameter was measured by the fluorescent probe Rhodamine 123 (R-123). The uptake of this dye is increased upon PHA stimulationz8 and depends also on the actual membrane potential of m i t o ~ h o n d r i a .FIGURE ~~ 3 reports the fluorescence histograms of young and old (from the same donor labeled with the asterisk in FIG.2) lymphocytes cultured for 48 hours in the presence (open histograms) and absence (shaded histograms) of PHA and analyzed on the same day. The cells from the young donors showed a
TABLE 2. 3H-Thymidine Incorporation in Lymphocytes Expressing c-myc and c-myb Proteins cpm/c-myc+ Cells Young Old
3,752 2,556
2 2
261 177
Difference - 38%
cpm/c-myb' Cells 4,318 3,368
2 2
246 213
Difference - 22%
NOTE: Entries are the mean 2 SEM of 10 experiments. The value for each individual was calculated by dividing the result of 'H-thymidine incorporation by the percentage of cells expressing the c-myc- and c-myb-encoded proteins.
strong increase of R-123 fluorescence due to PHA stimulation, and 78% of the lymphocytes showed a mean fluorescence between channels 110 and 150. On the contrary, only 30% of the cells from the old donors were able to reach the same level of fluorescence. Another small population (about 25%) of cells was able to increase the uptake of R-123 upon stimulation, but to a lower extent as compared to the young lymphocytes. Thus for these samples from old subjects we found an age-dependent alteration; however, it remains to be disclosed whether the reduced proliferation induced by PHA might be explained by impairments at the mitochondrial level and/or by the signal-transducing pathways responsible for mitochondria1 activation.
CONCLUSIONS Taken together our data support the view that the age-dependent decline of the lymphocyte response to mitogens can be accounted for by impairments at different levels along the time-dependent events that regulate the cell cycle. The reduced responsiveness of the old lymphocytes to depolarizing and repolar-
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izing conditions is a clear sign of an impairment of ion-channel functionality that may be involved in the alteration of physicochemical properties of aged plasma membranes. Although the role of the ion channel in cell proliferation is not completely understood, it is of importance that ion channel blockers are able to inhibit lymphocyte pr~liferation.~~
Log of fluorescence in arbitrary units (channel N”) FIGURE 3. R-123 fluorescence histograms of lymphocytes from young (A) and old (B) donors. Shadedand open histograms represent the control and PHA-stimulatedcells after 48 hours of incubation at 37°C in 5% C 0 2atmosphere, respectively. The cells with a fluorescence signal between channels 110 and 150 were taken to be cells with highly activated mitochondria.
Due to the scarcity of the lymphocytes obtained from each old individual, we have not been able to perform membrane potential measurements on cells from the same donors for which other parameters were measured. However, it is likely that the impairment at the level of ion channel functionality may play a role in the alterations observed for the other parameters. Experiments are in progress to verify this hypothesis.
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The reduced uptake of MC 540 observed in samples from the same old subjects suggests another type of impairment at the level of early activation processes and, for these donors, may also account for the modifications found thereafter. In the present work we followed the progression along the cell cycle of lymphocytes, analyzing c-myc and c-myb expression as markers of the G,/GI and G , / S phase, respectively. An age-dependent decrease of the percentage of cells expressing the protein encoded by these two genes was observed. What is of particular importance is that not all the cells able to express the c-myb proteins were able to synthesize DNA, as judged by the ratios reported in TABLE2. This suggests that the expression of the c-myb protein is a necessary3'.'* but not sufficient condition for the old cells to enter the S phase. This fact is not surprising in light of the complexity of cell pr~liferation'~ and the lack of information on the links between the expression of the different molecules. In this context, we showed an interesting example, in which a possible cause of the impairment of the age-related proliferative response may be studied at the level of the mitochondria or of the processes that lead to mitochondrial activation.
SUMMARY Several parameters generally believed to be necessary for the activation and progression of proliferation of human lymphocytes have been investigated and compared with special reference to aging. The responding capacity of plasma membrane potential to depolarizing and also repolarizing conditions induced by exposure to mitogens like PHA was lower in lymphocytes from old donors as compared to those of young ones. This indicates a significant age-dependent difference in the readiness to respond to channelactivating perturbations. As an early signal of activation, after one hour PHA stimulation the merocyanine 540 uptake by the lipid regions was chosen, based on the property of this fluorescent probe to bind to loosely packed lipids of the plasma membrane. The proteins encoded by the c-myc and c-myb genes were chosen as markers of the G,/G, and G , / S phased transition, respectively. The mean number of cells that increased the uptake of MC 540 following mitogenic stimulation did not differ in young vs. old individuals. However, 4 samples out of 10 from the old population showed lower MC 540 fluorescence than the lowest signal from the young population. The number of responding cells was decreased during aging when the presence of the c-myc protein was taken as its measure; and this decrease was further accentuated, determining the expression of the c-myb protein. This frequently encountered age-dependent pattern, however, was not followed by the lymphocytes of all old donors. One example is reported in which the MC 540 uptake, the c-myc and c-myb expression in the cells from one old subject fell in the range of the young subjects. However, even in this case, the response of the lymphocytes as measured by 3H-thymidine incorporation was only 64% of that of young subjects. For this sample, we found an impairment of the response at the mitochondrial level. In addition to these parameters, the amount of 3H-thymidine incorporated by the cells expressing the c-myb protein was calculated. The values in old individuals were lower than those in the young, suggesting that not all the cells expressing the c-myb protein were able to synthesize DNA in lymphocyte populations from the elderly. Our data support the view that the age-dependent decline of lymphocyte
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responsiveness to mitogens can be accounted for by impairments at different levels. Together with the most frequently encountered age-dependent modifications, such as the activation of the c-myc and c-myb genes shown in this paper, similar phenomena have been observed by others for interleukin (IL)-2 synthesis and IL-2 receptor expression indicating that there are some other alterations that may also play a significant role in blocking the proliferation of old lymphocytes.
ACKNOWLEDGMENTS The authors thank Mr. G. Mazzarini and R. Pierandrei for their technical assistance. REFERENCES 1.
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6. 7.
8. 9.
10.
11.
12. 13.
PISCIOTTA, A. B., D. W. WESTRING, C. DEPREY& B. WALSH.1967. Mitogenic effect of phytohemagglutinin at different ages. Nature 215: 193- 194. GILBERTSON, V. A. & E. J. YUNIS.1973. Lymphocyte phytohemagglutinin responsiveness, immunoglobulins and auto-antibodies in aging humans. J. Immunol. 111: 1101-1 107. WEKSLER,M. E. & T. M. HUTTEROTH.1974. Impaired lymphocyte function in aged humans. J. Clin. Invest. 53: 99-104. STAIANO-COICO, L., Z. DARZYNKIEWICZ, M. R. MELAMED & M. E. WEKSLER.1984. Impaired proliferation of T lymphocytes detected in elderly humans by flow cytometry. J. Immunol. 132: 1788-1792. MURASKO, D. M., P. WEINER& D. KAYE.1987. Decline in mitogen induced proliferation of lymphocytes with increasing age. Clin. Exp. Immunol. 70: 440-448. RIVNAY, B., S. BERGMAN, M. SHINITZKY & A. GLOBERSON. 1980. Correlation between membrane viscosity serum cholesterol, lymphocyte activation and aging in man. Mech. Ageing Dev. 12: 119-126. R. RECCHIONI, F. MORONI,M. FALASCA & L.PIANTAPIERI,C., F. MARCHESELLI, NELLI. 1990. Food restriction in female Wistar rats: I. Survival characteristic, membrane microviscosity and proliferative response in lymphocytes. Arch. Gerontol. Geriatr. 11: 99-108. LUSTYIK, G., H. M. HALLGREN, N. BERGH& J. J. O'LEARLY.1990. Effect of preliminary culture on the membrane microviscosity of lymphocytes from young and old donors. Arch. Gerontol. Geriatr. 1 0 77-88. WHISLER, R. L., I. G. NEWHOUSE, R. L. DONNERBERG & C. M. TOBIN. 1991. Characterization of intracellular ionized calcium responsiveness and inositol phosphate production among resting and stimulated peripheral blood T cells from elderly humans. Aging Immunol. Inf. Dis. 3: 27-36. GAMBLE,D. A., R. SCHWAB, M. E. WEKSLER & P. SZABO. 1990. Decreased steady state c-myc m-RNA in activated T cell cultures from old humans is caused by a smaller proportion of T cells that transcribe the c-myc gene. J. Immunol. 144: 3569-3573. DEGUCHI, Y.,S. NEGORO,H. HAM, S. NISHIO& S. KISHIMOTO.1988. Age-related changes of proliferative response, kinetics of expression of protooncogenes after the mitogenic stimulation and methylation level of the protooncogene in purified human lymphocytes subsets. Mech. Ageing Dev. 44: 153-168. TOLLEFSBOL, T. 0. & H. J. COHEN.1986. Expression of intracellular biochemical defects of lymphocytes in aging: Proposal of a general aging mechanism which is not cell-specific. Exp. Gerontol. 21: 129-148. G. R. 1989. Contingent genetic regulatory events in T lymphocyte activaCRABTREE, tion. Science 243: 355-361.
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14. THOMAN, M. L. & W. 0. WEIGLE.1989. The cellular and subcellular bases of immunosenescence. Adv. Immunol. 46: 221-261. 15. CHOPRA,R. K . 1990. Mechanism of impaired T-cell function in the elderly. Rev. Biol. Res. Aging 4: 83-104. S. & S. J. DIXON.1989. Ion transport, membrane potential and cytoplasmic 16. GRINSTEIN, pH in lymphocytes: Changes during activation. Physiol. Rev. 69: 417-481. A., J. A. LEDBETTER & P. S. RABINOVITCH. 1988. Reduced proliferation 17. GROSSMAN, of T lymphocytes in aged humans is predominantly in the CD8+ subset and is unrelated to defects in transmembrane signaling which are predominantly in the CD4' subset. Exp. Cell Res. 180: 367-382. G . & J. J. O'LEARLY.1989. Aging and mobilization of intracellular calcium 18. LUSTYIK, by phytohemagglutinin in human T cells. J. Gerontol. 44: 30-36. F. MORONI,L. BALKAY, T. MARIAN,L. TRON& S. DAMJA19. PIERI,C., R. RECCHIONI, 1989. Voltage and ligand gated sodium channels may regulate pump activity NOVICH. in human, mouse and rat lymphocytes. Biochem. Biophys. Res. Commun. 160: 999-1002. R. RECCHIONI, F. MARCHESELLI, M. FALASCA. R. ANTONICELLI 20. PIERI,C..F. MORONI, & S. DAMJANOVICH. 1992. Aging impairs membrane potential responsiveness as well as opening of voltage and ligand gated Na+ channels in human lymphocytes. Arch. Gerontol. Geriatr. 14 145-154. E. W., R. K. CHEUNG & S. GRINSTEIN. 1984. Role of membrane potential 21. GELFAND, in the regulation of lectin induced calcium uptake. J. Cell. Physiol. l21: 533-539. E. W., A. TORDAI& G. GARDOS.1990. Membrane potential selectively 22. LARDAKY. inhibits receptor operated calcium channels in human T (Jurkat) lymphoblast. Biochim. Biophys. Acta 1027: 130-140. L., C. PIERI.R. RECCHIONI, F. MORONI, L. BENE,L. TRON& S. DAMJANOV23. MATYUS, ICH. 1990. Voltage gating of Ca'+-activated potassium channels in human lymphocytes. Biochem. Biophys. Rec. Commun. 171: 325-329. P., K. MATTOCKS & R. A. SCHLEGEL. 1983. Merocyanine 540 a fluores24. WILLIAMSON, cent probe sensitive to lipid packing. Biochim. Biophys. Acta 732: 387-393. & R. A. SCHLEGEL. 1986. Alteration in plasma 25. DELBUONO,B. J.. P. L. WILLIAMSON membrane lipid organization during lymphocyte differentiation. J. Cell. Physiol. 126: 379-388. A. M., T. J. BRACIALE & V. L. BRACIALE. 1989. RegulationofTlymphocyte 26. CHURILLA, proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state. J. Exp. Med. 170: 105-121. R. RECCHIONI & F. MORONI.1991. Mitochondria1 mem27. PIERI,C.,F. MARCHESELLI, brane mass and membrane potential of PHA stimulated human lymphocytes during aging. In preparation. Z., L. STAIANO-COICO & M. R. MELAMED.1981. Increased mito28. DARZYNKIERWICZ, chondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. USA 78: 2383-2387. L . V., M. L. WALSH,B. J. BOCKUS& L. B. CHEN.1981. Monitoring relative 29. JOHNSON, mitochondria1 membrane potential in living cells by fluorescence microscopy. J. Cell. Biol. 88: 526-535. M. D. CAHALAN & S. GUPTA. 1984. Voltage30. CHANDY,K . G., T. E. DE COURSEY, gated potassium channels are required for human T-lymphocyte activation. J. Exp. Med. 160: 369-385. R., G. SCHWAB, E. WICKSTROM, S. LOONGLOKE,D. H. PLUZNIK, R. WATT 31. HEIKKILA, & L. M. NECKERS.1987. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from Go to G , . Nature 328: 445-449. G. P., Z. S. BRELVI,S. C. FELDMAN & R. A. WATT. 1986. Participation 32. STUDZINSKI, of c-myc protein in DNA synthesis of human cells. Science u4: 467-470.
INTRODUCTION It has long been recognized that the responsiveness of lymphocytes to mitogenic stimulation is impaired in elderly human patients.'-6 Several hypotheses have been proposed to explain the mechanism of such an impairment. The increased microviscosity of the membrane lipid domain may be (at least partially) responsible for the age-dependent decrease of the lymphocyte re~ponse.~.' Support for this hypothesis stems from the observation that preculturing the lymphocytes from old donors for 48 hours in culture medium containing 10% human serum resulted in a decrease of membrane microviscosity and an increase of the response to PHA.8 Other authors pointed out that the impairment was at the level of early signal tran~duction,~ genomic activation,I0.I1or protein synthesis.I2 The response of lymphocytes to mitogenic or alloantigenic stimulation is very complex. More than 70 molecules are specifically regulated during the time kinetics of this p r o c e ~ s . In ' ~ principle, any impairment of such a regulatory step may have a causative relationship with the age-dependent decrease of the response observed in human lymphocytes. Although the age-related decrease of many parameters has been well documented (see for review Refs. 14, 15), few studies have examined the individual changes and the correlations between different parameters in each individual. In the present work, four different parameters have been compared that reflect different stages in the progression of lymphocyte activation-namely , the modification of cell membrane structure, the c-myc and c-myb expression, and the incorporation of labeled thymidine into newly synthesized DNA. In addition, data are presented on the responsiveness of the membrane potential of peripheral blood lymphocytes to depolarizing conditions as well as to PHA treatment.
a
This work was supported in part by a grant to C.P. from the National Research Council. 110
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MEMBRANE POTENTIAL STUDIES The binding of alloantigens or mitogenic lectin to the lymphocyte plasma membrane results in, among other things, changes of ion channel activities and of membrane potential. l6 The increase of intracellular free Ca+ induced by mitogens has already been extensively investigated: however, conflicting data have been reported on the age dependency of this parameter in h ~ m a n s . ~ . ~ 'As - ' *regards the alteration of membrane potential, a repolarizing effect of PHA treatment has been demonstrated,16 but data on the effect of aging on this parameter are lacking. Recently, we demonstrated the presence of ligand- and voltage-gated Na' channels in human lymphocytes using bretylium tosylate (BT), a quaternary ammoniumion. as ligand. BT treatment resulted in an increase of intracellular Na' content, with a consequent activation of Na'-K+-ATPase pump and repolan~ation.'~ This sensitive mechanism was found to be impaired by aging.?O In the present paper we report that the extent of PHA-induced repolarization of slightly depolarized lymphocytes depends on the age of the donors. To measure the membrane potential changes, the fluorescent probe oxonol [bis ( I ,3, dibutylbarbituric acid-(S)-trimethilene oxonol)] was applied, as described in previous papers.19-?()It has to be noted that the repolarization of lymphocytes with PHA is more complex than that induced by BT. Indeed, although complete depolarization inhibits the Ca' influx pathway,?' it was not able to influence mitogen-induced internal calcium release.?? Among conditions that partially depolarize cells, an increase of free intracellular Ca' concentration occurs (see in Ref. 22: we also have similar data not shown here) subsequent to mitogenic stimulation. Thus, at least two mechanisms may be responsible for the PHA-induced repolarization. According to our model, one of these mechanisms is the opening of voltageand ligand- (mitogen) gated Na+ channels and the activation thereby of Na+/K'dependent ATP-ase: the second mechanism is the opening of Ca'-activated K' channels, which also proved to be potential sensitive in lymphocytes.23 FIGURE 1 shows a typical example of the repolarizing effect of PHA in human lymphocytes from young (A) and old (B) persons. The increase or the decrease of fluorescence (channel number) is indicative of depolarization and repolarization, respectively. The bis-oxonol that provides the fluorescence signal is negatively charged: thus the fluorescence intensity and membrane potential of the cell have an inverse relationship. The shaded histograms represent the cells depolarized by increasing the extracellular K + from 5 to 73 mM, an isosmotic mechanism. Since the basal resting membrane potential (i.e., the fluorescence measured at 5 mM extracellular K') is not different in cells from young and old subjects (see Ref. 20; we have similar data not shown here), it is easy to conclude that the response of the old cells to the depolarizing conditions is lower than that of young ones. Although the membrane permeability could be an important factor in this phenomenon, a more complex mechanism cannot yet be excluded. Addition of PHA to the depolarized cells resulted in a repolarization (open histograms), the extent of which is again lower in old (B) as compared to young lymphocytes (A). TABLE I summarizes the results of the analysis of lymphocytes from 10 young and 10 old persons. The positive figures indicate a shift of mean fluorescence of the oxonol to the right (depolarization) and the negative figures the extent of the shift to the left (repolarization). Both the responses are impaired by aging, which supports the idea that they were initiated by an alteration of ion channel activities. Thus, aging may impair the response of lymphocytes to mitogens at a very early phase of the activation process. +
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Fluorescence in arbitrary units [channel No) FIGURE 1. Oxonol fluorescence histograms of lymphocytes from young (A) and old (B) subjects. Shaded and open histograms represent the cells depolarized by isosmotic increase of extracellular K + (73 mM) and subsequent incubation with PHA for five minutes at 37"C, respectively.
TABLE 1. Shift of Mean Oxonol Fluorescence in Young and Old Lymphocytes Youne K5-K73 K73-PHA
+48.5 -33.8
2 f
Old 1.4 2.8
+38.2 -20.5
If. f
3.0 2.6
p p
< 0.001 < 0.001
NOTE: Entries are mean If. SEM of 10 experiments. K5-K73 indicates the increase of mean oxonol fluorescence (depolarization) upon the isotonic increase of extracellular K+ concentration from 5 mM to 73 mM. K73-PHA indicates the decrease of mean oxonol fluorescence subsequent to PHA addition to the culture medium containing 73 mM K + as in FIG. I.
1W
PIER1 et a/.: RESPONSE TO PHYTOHEMAGGLUTININ
ACTIVATION OF CELL MEMBRANE AND GENOMIC LEVELS As another early event, the binding of the stimulatory ligand to the lymphocytes’ plasma membrane leads to well-defined structural changes. We utilized these changes, occurring in the lipid packing, to monitor the percentage of lymphocytes activated by I-hour exposure to PHA at 37°C. For this purpose the uptake of merocyanine 540 (MC 5401, a fluorescent probe that preferentially binds to disordered membranes, was used.24-25 In addition, we determined the quantity of proteins, encoded by the activated c-myc and c-myb protooncogenes, which can also serve as markers of Go/G,and G,,/S probe transition, respectively. Taking these three measured parameters together, they provide information on the progression of cell activation processes for each individual cell. The results are presented in FIGURE 2. Since they were able to increase the amount of MC 540 bound to their membranes (i.e., the percentage of activated cells), the lymphocytes had a wider
OLD
YOUNG
1
O’
50 -
60
-ln
:
40:
e 0 &
c
30-
:
20-
I
a
=
. 10-
n,
FIGURE 2. Percent of the cells from young and old individuals activated by PHA stimulation as measured by MC 540 uptake and by c-myc- and c-myb-encoded protein expression.
distribution in old samples (range 22-54, mean 2 SEM 38.0 2 3.7) than in young samples (range 34-52, mean SEM 45.4 1.9). Although the difference between the means was not statistically significant, it is worthy of note that in 4 out of 10 old samples the percentage of activated lymphocytes was lower than the lowest percentage for the young samples. This suggests a delay of the response and/or an impairment of the mechanisms leading to the modification of membrane lipid packing for these samples. Examining a higher number of samples is likely to yield statistically significant differences. The presence of the c-myc-coded protein was assessed by indirect immunofluorescence, determined 16 hours after PHA stimulation. Rabbit polyclonal antibodies against a bacterially expressed protein encoded by exon, of the human c-myc gene (Medac, Molecular Biology, Germany) was used as first antibody. Fluoresceinated goat anti-rabbit IgG was the second antibody. In general, in the young samples the percentage of cells showing positivity for
*
*
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c-myc protein expression was higher than that measured with MC 540, suggesting that an increasing number of lymphocytes was activated by PHA later than one hour after exposure. On the contrary, in the lymphocytes from old donors, only 3 out of 10 samples showed a higher percentage of cells expressing c-myc than those measured by MC 540 incorporation. In the other samples, a more-or-less expressed decrease in the amount of positive cells was observed. It is interesting to note that in half of the samples from old individuals the percentage of cmyc-positive cells fell in the range that was characteristic for cells from the young. However, if one considers the whole population, the difference between the means was statistically significant (mean 2 SEM 52.2 f 2.2 in the young vs. 37.3 f 4.4 in the old, p < 0.01). The general meaning of these data is that the activation of the c-myc gene andlor the consequent synthesis of the specific protein have been impaired by aging; this is in agreement also with other authors.IoJ1However, this alteration is not an all-or-nothing phenomenon. As regards the number of cells expressing the c-myb-encoded protein, this parameter was determined 66 hours after PHA stimulation by indirect immunofluorescence, using as first antibody a polyclonal rabbit antibody to human c-myb protein (Medac, Molecular Biology, Germany). Fluoresceinated goat anti-rabbit IgG was the second antibody. As shown in FIGURE 2, not all the lymphocytes expressing c-myc protein were able to express also the c-myb-encoded one, and the decrease in the percentage of cells that lost this capability was higher in the old ( - 10.2%) than in young sample (-6.5%). Interestingly enough, a strong decrease in the number of cells positive for c-myb was observed from young to old populations (mean k SEM 45.7 f 2.5 and 27.1 f 3.2, in young and old lymphocytes, respectively; p < 0.001). A likely explanation for this decrease can be found at the level of regulation. It is well established, indeed, that c-myb gene expression is preceded by the binding of IL-2 to its receptor,26and both IL-2 synthesis and high-affinity IL-2 receptor expression seem to be impaired during aging (for review see Ref. 15).
'H-THYMIDINE INCORPORATION AND ITS CORRELATION WITH C-MYC AND C-MYB PROTEIN EXPRESSION An important question to be answered is whether the observed decrease in cmyc and c-myb expression could account for the decreased DNA synthesis observed in stimulated lymphocytes during aging. In order to answer this question we calculated the amount of 3H-thymidine incorporated by the cells expressing the c-myc- and c-myb-encoded proteins. The results are shown in TABLE2. The difference between the young and old populations observed for these parameters when the c-myc-positive cells were taken into account can easily been explained by the age-dependent decrease of a number of factors, such as IL-2 receptor expression, 11-2 synthesis (for review see Ref. 15), and c-myb activation (present paper), which follow c-myc activation and precede and are necessary for DNA synthesis. We observed that a difference still existed between young and old cells in the amount of labeled thymidine incorporated into the newly synthesized DNA even in the case of c-myb-positive cells. This finding suggested that despite the presence of the c-myb protein, not all of the cells from old donors are able to progress from G I to S phase, because of impairment(s) at level(s) different from those taken into account in the present paper. On the other hand, considering the lymphocytes from the old subject labeled with the asterisk in FIGURE 2, all the
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parameters analyzed fell in the ranges characteristic of the young population. Thus we could not explain the reduced 3H-thymidine uptake of this old sample as compared to the young one analyzed on the same day (125 x lo3cpm vs. 194 x lo3 cpm, respectively). A PARTICULAR CASE OF AGE-DEPENDENT ALTERATION OF
CELLULAR PARAMETERS Apart from the data presented above, another parameter has been analyzed in the lymphocytes of the same young and old subjects (mitochondrial activation and its results will be reported in detail elsewhere2'). This parameter was measured by the fluorescent probe Rhodamine 123 (R-123). The uptake of this dye is increased upon PHA stimulationz8 and depends also on the actual membrane potential of m i t o ~ h o n d r i a .FIGURE ~~ 3 reports the fluorescence histograms of young and old (from the same donor labeled with the asterisk in FIG.2) lymphocytes cultured for 48 hours in the presence (open histograms) and absence (shaded histograms) of PHA and analyzed on the same day. The cells from the young donors showed a
TABLE 2. 3H-Thymidine Incorporation in Lymphocytes Expressing c-myc and c-myb Proteins cpm/c-myc+ Cells Young Old
3,752 2,556
2 2
261 177
Difference - 38%
cpm/c-myb' Cells 4,318 3,368
2 2
246 213
Difference - 22%
NOTE: Entries are the mean 2 SEM of 10 experiments. The value for each individual was calculated by dividing the result of 'H-thymidine incorporation by the percentage of cells expressing the c-myc- and c-myb-encoded proteins.
strong increase of R-123 fluorescence due to PHA stimulation, and 78% of the lymphocytes showed a mean fluorescence between channels 110 and 150. On the contrary, only 30% of the cells from the old donors were able to reach the same level of fluorescence. Another small population (about 25%) of cells was able to increase the uptake of R-123 upon stimulation, but to a lower extent as compared to the young lymphocytes. Thus for these samples from old subjects we found an age-dependent alteration; however, it remains to be disclosed whether the reduced proliferation induced by PHA might be explained by impairments at the mitochondrial level and/or by the signal-transducing pathways responsible for mitochondria1 activation.
CONCLUSIONS Taken together our data support the view that the age-dependent decline of the lymphocyte response to mitogens can be accounted for by impairments at different levels along the time-dependent events that regulate the cell cycle. The reduced responsiveness of the old lymphocytes to depolarizing and repolar-
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izing conditions is a clear sign of an impairment of ion-channel functionality that may be involved in the alteration of physicochemical properties of aged plasma membranes. Although the role of the ion channel in cell proliferation is not completely understood, it is of importance that ion channel blockers are able to inhibit lymphocyte pr~liferation.~~
Log of fluorescence in arbitrary units (channel N”) FIGURE 3. R-123 fluorescence histograms of lymphocytes from young (A) and old (B) donors. Shadedand open histograms represent the control and PHA-stimulatedcells after 48 hours of incubation at 37°C in 5% C 0 2atmosphere, respectively. The cells with a fluorescence signal between channels 110 and 150 were taken to be cells with highly activated mitochondria.
Due to the scarcity of the lymphocytes obtained from each old individual, we have not been able to perform membrane potential measurements on cells from the same donors for which other parameters were measured. However, it is likely that the impairment at the level of ion channel functionality may play a role in the alterations observed for the other parameters. Experiments are in progress to verify this hypothesis.
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The reduced uptake of MC 540 observed in samples from the same old subjects suggests another type of impairment at the level of early activation processes and, for these donors, may also account for the modifications found thereafter. In the present work we followed the progression along the cell cycle of lymphocytes, analyzing c-myc and c-myb expression as markers of the G,/GI and G , / S phase, respectively. An age-dependent decrease of the percentage of cells expressing the protein encoded by these two genes was observed. What is of particular importance is that not all the cells able to express the c-myb proteins were able to synthesize DNA, as judged by the ratios reported in TABLE2. This suggests that the expression of the c-myb protein is a necessary3'.'* but not sufficient condition for the old cells to enter the S phase. This fact is not surprising in light of the complexity of cell pr~liferation'~ and the lack of information on the links between the expression of the different molecules. In this context, we showed an interesting example, in which a possible cause of the impairment of the age-related proliferative response may be studied at the level of the mitochondria or of the processes that lead to mitochondrial activation.
SUMMARY Several parameters generally believed to be necessary for the activation and progression of proliferation of human lymphocytes have been investigated and compared with special reference to aging. The responding capacity of plasma membrane potential to depolarizing and also repolarizing conditions induced by exposure to mitogens like PHA was lower in lymphocytes from old donors as compared to those of young ones. This indicates a significant age-dependent difference in the readiness to respond to channelactivating perturbations. As an early signal of activation, after one hour PHA stimulation the merocyanine 540 uptake by the lipid regions was chosen, based on the property of this fluorescent probe to bind to loosely packed lipids of the plasma membrane. The proteins encoded by the c-myc and c-myb genes were chosen as markers of the G,/G, and G , / S phased transition, respectively. The mean number of cells that increased the uptake of MC 540 following mitogenic stimulation did not differ in young vs. old individuals. However, 4 samples out of 10 from the old population showed lower MC 540 fluorescence than the lowest signal from the young population. The number of responding cells was decreased during aging when the presence of the c-myc protein was taken as its measure; and this decrease was further accentuated, determining the expression of the c-myb protein. This frequently encountered age-dependent pattern, however, was not followed by the lymphocytes of all old donors. One example is reported in which the MC 540 uptake, the c-myc and c-myb expression in the cells from one old subject fell in the range of the young subjects. However, even in this case, the response of the lymphocytes as measured by 3H-thymidine incorporation was only 64% of that of young subjects. For this sample, we found an impairment of the response at the mitochondrial level. In addition to these parameters, the amount of 3H-thymidine incorporated by the cells expressing the c-myb protein was calculated. The values in old individuals were lower than those in the young, suggesting that not all the cells expressing the c-myb protein were able to synthesize DNA in lymphocyte populations from the elderly. Our data support the view that the age-dependent decline of lymphocyte
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responsiveness to mitogens can be accounted for by impairments at different levels. Together with the most frequently encountered age-dependent modifications, such as the activation of the c-myc and c-myb genes shown in this paper, similar phenomena have been observed by others for interleukin (IL)-2 synthesis and IL-2 receptor expression indicating that there are some other alterations that may also play a significant role in blocking the proliferation of old lymphocytes.
ACKNOWLEDGMENTS The authors thank Mr. G. Mazzarini and R. Pierandrei for their technical assistance. REFERENCES 1.
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11.
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