Experimental Section Gerontology 1992;38:301-307
McGill University School of Medicine, Thorp Laboratories of Clinical Immunology, Royal Victoria Hospital, Montreal, Que., Canada
Keywords Immunology Aging AMLR Alzheimer’s
Immune Reactivity in Aging Autologous Mixed Lymphocyte Responses
Abstract The level of activity in the autologous mixed lymphocyte reac tion (AMLR) was studied in populations of young and old sub jects. AMLR activity was reduced in the older age group. A subpopulation of the older age group who had Alzheimer’s dis ease was shown to have the lowest AMLR values. Within this group, those with a history indicating a more rapid develop ment of CNS-related disability from senescence showed the weakest AMLR responses. The AMLR values correlated nei ther with sex. nutritional status nor history of infections. This impairment of a central regulating immune reaction may be a significant variable in manifestations of the aging process.
Introduction Many of the functional elements of the immune system show some decrease in activ ity with aging [1-3]. This decline is accompa nied by an increased incidence of diseases associated with impairment of immune regu latory and host defense functions [3], Because of the key importance of the immune system in the overall body economy, theories have been developed to explain the aging process through the effects of progressive immuno logic defects [4, 5]. Individual components of the functioning immune system may decline at different
This work was supported through grants from the Canadian Geriatric Society and the Lucie and Ronald Javitch Fund.
Accepted: March 21,1992
rates. Thus, an immune regulation distur bance may result when only the T-helper or T-suppressor inducer subsets are involved [6, 7], Aging patients may develop serum autoan tibodies or a monoclonal gammopathy in the absence of an underlying autoimmune disease or plasma cell dyscrasia. The elucidation of the mechanisms and the phenomena that are involved in the agerelated changes of the immune system might lead to an appreciation of possible therapeutic approaches aimed at correcting some of these, and thereby preventing the negative sequelae on the welfare of the host.
C.K. Osterland Thorp Professor of Medicine c/o Royal Victoria Hospital 687 Pine Avenue West Montreal.Que. H3A 1A1 (Canada)
© 1992 S. Kargcr AG. Basel 0304-324X/92/ 0386-030152.75/0
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C.K. Osterland E.A. St. Louis
302
Osterland/St. Louis
Materials and Methods Patients and Subjects Three groups of patients were studied. The first consisted of young normal subjects with a mean age of 27 (range 22-36 years). The second was a population of ’normal’ aged individuals, mean age 73 (range 6774 years). These individuals exhibited a normal aging pattern and no evidence of active disease that could influence the results of laboratory studies. The third population group was hospitalized for age-related dis ability and suffered from Alzheimer’s disease (mean age 74, range 50-86 years). Blood sampling was made on a voluntary basis, with informed consent. In the case of individuals in the Alzheimer’s population, blood was acquired with family consent and was done at the same time as the patient was having venipunc ture for other diagnostic testing ordered by the attend ing physicians. The studies were approved by the Ethics Review Committee of the institution. Selection of study individuals was made with care taken to exclude any with active illnesses or those who were receiving medications that could influence the test results. The exclusion criteria used were similar to those described by Ligthart et al. [27] in the Senieur protocol. This meant that patients with infections, malignancy and malnutrition were excluded. Labora tory evidence of hypoproteincmia, renal failure, marked anemia or lymphopenia was also a basis for patient exclusion. Patients who were receiving atarac tic or antidepressive medications that may have had minor effects on in vitro immune function had the medication withdrawn 24 h prior to blood sampling. A review of the patients’ charts noted primary and secondary diagnoses. For the Alzheimer’s disease study group, a clinical judgement was made regarding two additional aspects of the patients’ disability: the first involved a global assessment of the severity of the clinical disability; the second noted the rapidity or pace with which the disability symptoms had pro gressed, from the time that symptoms were first docu mented until the time of study. A designation o f’rapid’ was assigned when the patient status had deteriorated to full disability within 6 months, and as ’slow'’ when 1 year or more elapsed between symptom onset and the occurrence of marked disability. These estimates were made independently by two observers with agreement in the classification. The patients’ nutritional status was noted objec tively and in terms of the dietary intake as indicated in the hospital chart. The frequency and nature of all doc umented infectious episodes occurring over the pre ceding 12 months were also tabulated.
AMLR in Aging
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The autologous mixed lymphocyte reac tion (AMLR) has been looked upon as having some importance in determining the func tional state of the immune system [4, 5]. The AMLR provides an in vitro means of cellular communication that stimulates the genera tion of cytokines and effector cells, in a non antigen-dependent fashion [4, 5. 8-10], In the AMLR assay, CD4+ T cells give a prolifera tive response to autologous MHC class II anti gens in the absence of known processed pep tide antigen [11-14], The presence of exoge nous antigens in the in vitro test system [15] does not explain the reactivity. While the dis play of processed specific peptide, derived from autologous or exogenous sources in the MHC class II groove is crucial to T-cell activ ity and the immune response, the AMLR reaction is predominantly dependent on the MHC class II alone. The AMLR involves the activation of greater numbers of T cells than is seen in T-cell response to a conventional peptide-MHC complex antigen. In this re gard, it is more similar to an allogeneic re sponse [5, 8. 16-22]. The activation of autoreactive T cells with specificity for self-MHC may represent a means by which self- versus non-self-discrimination and autoimmunity are regulated [ 17], The AMLR has been studied in the normal aging population as well as in many disease states [23-26], While most studies indicate a reduced activity and generation of effector cells, there has not been complete agreement. Fournier and Charriere [24] noted an increase in AMLR responses in the female subjects of their aging population study. In the current study, we carried out AMLR studies on a normal-aged population, an Alz heimer’s group and a group of young normal subjects. Reduction in AMLR was noted in the older group and was even more striking in the Alzheimer’s patients.
Rheumatoid and Antinuclear Factor Measurements Two non-organ-specific autoantibody activities, both known to occur with increased frequency in the sera of older patients, were measured. The results were subsequently correlated with the AMLR experiments. The antinuclear factors (ANA) were done by an indi rect immunofluorescence technique using human epi thelial Hep-2 cells as substrate (Quantafluor, Kallestad Laboratories Inc.), sera were screened at a 1/40 dilu tion. This technique detects serum autoantibodies spe cific for several nuclear and cytoplasmic constituents. There were no positives in the normal control group, although the test can be positive in less than 2.5% of a blood donor population. Rheumatoid factor (RF) analysis was by a standard immunochemical latex agglutination reaction (Rapi Tex RF, Behringwerke AG). Sera giving negative re sults in this assay contained no RF or had concentra tions < 20 IU/ml. Using this technique, the incidence of false positives in a normal healthy population is < 1%. As with the ANA autoantibody testing, there were no RF-positive individuals among the normal controls studied.
Results The AMLR results indicated differences among the population groups studied. The values for the aged population were lower
Table 1. AMLR results from a group of patients with Alzheimer’s disease and from two different age groups of normal controls Study group
n
Mean value ± SEM
Alzheimer’s 'Normal' aged ‘Normal’ young
24 9 11
4,876± 825 8,540 ±1,404 13,204 ±1,854
AMLR results are expressed as counts per minute 3H-thymidine incorporation minus the spearate back ground counts of B and T cells. AMLR was significantly (Mann-Whitney rank test) lower in Alzheimer’s patients than in either the normal aged (p < 0.05) or young (p < 0.01) groups. Differences in AMLR proliferative responses be tween the two normal groups failed to attain statistical significance at the p = 0.01 level.
than those of the younger, normal subjects (ta ble 1). Among the total aged population stud ied, those patients with a diagnosis of Alz heimer’s disease with related disability gave significantly lower values of 3H-thymidine in corporation than did ’controls’ in the similar age group not exhibiting Alzheimer’s demen tia or disability. Though the normal aged group had lower uptake values than the normal young group, the values did not reach statistical signifi cance (table 1). When all of the patients were grouped according to three defined age groups, there appeared to be a decrease in AMLR values that correlated with increasing age (fig. 1). Because some individual age pop ulations were poorly represented in the study, these results indicate only the trend of low ered AMLR with increasing age of these par ticular groupings. When the data were analyzed comparing male and female patients, no significant AMLR differences could be attributed to this variable (table 2).
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AMLR Heparinized blood samples were separated on a Ficoll-Hypaque (Pharmacia) gradient. Monocytes were removed by adherence to plastic dishes. T- and B-lymphocytc populations were obtained by E-rosetting the peripheral blood lymphocytes (PBL), then passing the mixture over another Ficoll-Hypaque gra dient. PBL could also be separated into T and non-T on a nylon wool column. The rosette-prepared lym phocytes and the nylon wool purified ones gave similar results. The responser T lymphocytes (1 X 105) were cultured with 1 X 105 stimulator B lymphocytes that had been inactivated with mitomycin C, 50 pg/ml (Flow Labs.). Cells were cultured in triplicate in U-bottom 96-well Linbro microtiter plates in RPMI 1640 supplemented with 10% human AB serum, 2 mM Lglutamine, 10 ntW Hepes buffer and containing 50 pg/ml gentamycin. Cultures were continued for 6 days, and then pulsed with 1 pCi 3H-thymidine for 16 h prior to harvesting and processing for counting.
n
Negative
cpm x 103
m Positive
3
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Fig. 1. AMLR proliferation (counts per minute 3Hthymidine incorporation - background counts of B and T cells) grouped according to age alone. Average proliferative responses in the individuals comprising the 60-69 and 70-79 year age categories were similar and were analyzed as a single 60-80 year age group. Although AMLR responses appeared to decrease with increasing age, the differences between groups were not statistically significant. Fig. 2. AMLR proliferation (expressed as counts per minute 3H-thymidine incorporation - background counts of B and T cells) in 23 patients with Alzheimer’s disease. Patients were subsequently grouped according to whether disease progression was judged gradual or rapid (see Materials and Methods). AMLR prolifera tive responses were lower in those patients who had experienced rapid degeneration (p < 0.05, Student’s t test). Fig. 3. AMLR proliferation (expressed as counts per minute 3H-thymidine incorporation) in 18 patients with Alzheimer’s disease. Patients were grouped ac cording to whether or not their sera tested positive for serum autoantibodies (see Materials and Methods). AMLR proliferative response were lower in the group whose sera contained detectable autoantibody activity (significant at 5% level, Mann-Whitney U test).
AMLR in Aging
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cpm x 103
f~~l Gradual I I Rapid
As described in Methods, the Alzheimer’s patients were grouped according to the pace at which each progressed clinically, in terms of their symptoms of senescence and disability. Patients were assigned either to a rapid or slow progression category. Those with whom this judgement could not be made with cer tainty were excluded. Figure 2 shows that the rapid progressors had significantly lower AMLR responses than the slow progressors. The age distribution was similar in these groups as was the overall senescence disability status at the time of examination. These sub jects represented the younger population group of table 1 and figure 1. The presence of the reduced AMLR values correlates posi tively (p < 0.05) with the presence of positive tests for the non-organ-specific autoantibod ies (fig. 3).
Table 2. AML R results grouped and analyzed according to sex
Alzheimer’s
‘Normal’ aged
‘Normal’ young
M n=7
F n= 17
M n=6
F n=3
M n=2
F n=9
4,524 (1,094)
5,049 (1,092)
8.683 (1.837)
8.255 (2,594)
16.769
12.417 (2.039)
Estimates of nutritional status of the hospi talized senescent patient group and the inci dence of infections over 1 year did not show any significant correlation with AMLR values or with the patient grouping as a whole. The nursing care received by these patients was attentive, and the dietary intake and nutri tional status of even the Alzheimer’s group appeared to be excellent. The frequency of infections was surprisingly low in all patient groups.
Discussion The AMLR response is a reaction of T lymphocytes against MHC class II related an tigens on B lymphocytes [21, 28], The reac tion occurs as a result of mixing an individu al’s T and B cells together in a ratio of about 1:1, thus exposing, in vitro, the patient's T lymphocytes to an unusually high frequency of Ia-antigen-positive B lymphocytes. The reaction is quite similar to the syngeneic mixed lymphocyte culture reaction in mice [4], While some studies have indicated that exogenous foreign antigens derived during cell separation procedures or culture condi tions play a major role in the response by gen erating an in vitro immune response [ 15], this is not a complete explanation for the phenom enon [5, 8, 16-22], The AMLR stimulation
response is not as vigorous as an allogeneic response by the same cell populations, but it is markedly greater than a response to individ ual antigen [29], Regardless of the sources of any antigen or antigenic peptides that might be associated with the MHC class II mole cules in the AMLR, the patient’s T cells pro duce a polyclonal response with the genera tion of effector cells. The primary responding cell is the CD4+ T-helper cell [12, 20, 30], It is typical, how ever, to see the generation of increased num bers of activated T-helper (CD4) and T-suppressor(CD8) lymphocytes [5, 13,20, 31 ] as a result of accompanying cytokine production, especially of interleukin-2. Secondary auto crine stimulation also occurs in the cell mix tures, causing CD8 and B-cell stimulation as well. In a physiological sense, the AMLR reac tion has been considered as one that may, in an in vivo form, exert some element of im mune regulation and immune response am plification because of its action of generating increased amounts of helper, suppressor and lymphocytotoxic cell (NK) activity [32], The AMLR responses have been found to be reduced in a variety of different medical conditions, especially those that affect the im mune system through autoimmunity or lym phoma [9, 12, 17, 25, 29, 33], In systemic lupus erythematosus patients showing re duced AMLR, there appears to be an impair
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Values represent counts per minute 3H-thymidine incorporation mi nus background counts of stimulators and responders. Values in parenthe ses are standard errors.
ment of the generation of suppressor cells in the reaction [ 12], a finding considered to be of some relevance to the course of lupus itself. Results from studies on the relationship of age and senescence in the AMLR response have been somewhat controversial. Fournier and Charriere [24] reported an increase in AMLR activity in older women. Most investi gators have reported a decrease in the activity regardless of sex [23. 25, 26, 34]. If AMLR activity does become reduced with age. its proposed physiological role in immune regu lation could play an important role in the occurrence and progression of the other known defects in immune function seen with aging, such as loss of suppressor-cell function, increased autoantibody formation and re duced effector cells with poor immune sur veillance mechanisms. The studies reported here support the pre vious findings of reduced AMLR activity in the aged population, with no differences noted between the sexes. It has been pointed out [27] that immunogerontologic studies re quire careful patient selection using a proto col similar to the Senieur grouping before ageor disease-related correlations can be made. A subpopulation of the aged suffering from de bilitating Alzheimer's was found to have the lowest AMLR values. It is not clear what this finding signifies, beyond the possibility that impairment of the immune system might be one of numerous factors that contribute to the acceleration and severity of the aging process.
It was of interest that patients judged to exhibit the most rapid pace of age-related symptom progression also showed the great est reduction in their AMLR responses. One of the most important goals for human aging studies is to develop strategics for min imizing the handicaps of old age and to find better therapies for preventing geriatric dis eases and disabilities. When one finds a com mon age-related change in 'normal subjects’, this does not indicate that the change is neces sarily harmless. It may act as a cofactor to damage, coming from other predisposing fac tors. Examples of such cofactors in aging might include the effects of an elevation in blood pressure or blood glucose values. Per haps it is most important to know what changes occur as a function of normal aging. It may be possible to correlate changes in these variables in subjects identified as exhibiting minimal, moderate or advanced aging charac teristics. Most age-related alterations in the activity parameter of a biological response are likely to show only a percentage change from normal. The signals identifying such changes, therefore, may be low in a quantitative sense yet be quite significant for the individual.
Acknowledgements The authors acknowledge the assistance of Ms. Elisa Monaco and Mrs. L. Warner in the preparation of the manuscript, and the staff of the Douglas Hospi tal in obtaining patient materials for study.
I Wade A. Szewczuk M: Aging, idiotvpe shifts, and compartmentalization of the mucosal associated lym phoid system: in Kunkel H. Dixon F (eds): Advances in Immunology. New York. Academic Press, 1985. vol 36.
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2 Szewczuk M. Wade A: Cellular aging, idiotype repertoire changes and mucosal-associated lymphoid system; in Sauer HW (ed): Cellular Ageing. Monogr Dev Biol. Basel. Karger. 1984, vol 17. pp 122-141.
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3 Makinodan T. Kay MMB: Age in fluences on the immune system. Adv Immunol 1980:29:287-330. 4 Wekslcr M. Kozak R: Lymphocyte transformation induced by autolo gous cells. J Exp Med 1977:146: 1833.
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References
16 Laffon A, Alcocer-Varela J, Alarcon-Segovia D: The AMLR is not primarily due to xenogeneic antigen stimulation. Clin Immunol Immunopathol 1983;28:304-308. 17 Romain PL, Lipsky P: Immuno globulin secretion during the autolo gous mixed lymphocyte reaction in man. J Immunol 1983; 120:1146— 1150. 18 Smolen JS, Luger T, Chused TM, Steinberg AD: Responder cells in the human autologous mixed lym phocyte reaction. J Clin Invest 1981;68:1601-1607. 19 Moody C, Gupta S. Weksler M: Lymphocyte transformation in duced by autologous cells. Xenoantigens are not required for autolo gous mixed lymphocyte reaction. J Clin Immunol 1983;3:100-103. 20 Palacios R. Anderson V: Autologous mixed lymphocyte helper and cyto toxic T-cell functions and increased proliferative response and induction of suppressor T cells. Cell Immunol 1982;66:88-98. 21 Palacios R, Claesson L, Moller G, Peterson P, Moller E: The alpha chain, not the beta chain, of HLADr antigens participates in the acti vation ofT cells in autologous MLR. Immunogenetics 1982;15:341-350. 22 Lattime EC, Gillis S. Pecoraro G, Stutman O: Ia-dcpendent interleu kin-2 production in syngeneic cellu lar interactions. J Immunol 1982; 128:480. 23 Moody C. Innes J, Coico L, Incefy G, Thaler H, Weksler M: The effect of age on AMLR. Immunology 1981;44:431-438. 24 Fournier C, Charriere J: AMLR in man - Relationship with age and sex. Cell Immunol 1981:60:212218. 25 Indiveri F, Pierre I, Viglione D. Pende D. Russo C. Pellegrino M, Ferrone S: Human T lymphocytes in aging and malignancy: Abnormali ties in PHA-induced la expression and in functional activity in autolo gous MLR. Cell Immunol 1983;76: 224-231. 26 Fernandez A. MacSween I: De creased autologous mixed lympho cyte reaction with aging. Mech Age ing Dev 1980;12:245-250.
27 Lighthart GJ, Corberand J, Four nier C, Galanaud P, Hymans W, Kenncs B, Müller-Hermclink HK, Steinman GG: Admission protocol for immunogerontological studies in man: The Senieur protocol. Mech Ageing Dev 1984:28:47. 28 Romain PL, Schlossman SF. Reinherz EL: Surface molecules involved in self recognition and T-cell activa tion in the autologous mixd lympho cyte reaction. J Immunol 1984:133: 1093-1100. 29 Moody CE, Casazza BA, Christen son WN, Weksler ME: Lymphocyte transformation induced by autolo gous cells. VIII. Impaired autolo gous mixed lymphocyte reactivity in patients with acute infectious mono nucleosis. J Exp Med 1979; 150: 1448. 30 Smolen JS, Chused TM, Novotny EA, Steinberg AD: The human au tologous mixed lymphocyte reac tion: Immune circuits. J Immunol 1982;129:1050-1055. 31 Yu D, Chiorazzi NC, Kunkel HG: Helper factors derived from autolo gous mixed lymphocyte cultures. Cell Immunol 1980:50:305-313. 32 Goto M. Zvaifler N: Characteriza tion of the killer cell generated in the autologous mixed lymphocyte reac tion. J Exp Med 1983; 157:1309— 1323. 33 Hahn B. Pletscher S, Muniain M. MacDermott R: Supprcsssion of AMLR by sera from SLE patients. Arthritis Rheum 1982:25:381. 34 Canonica GW, Ciprandi C, Caria M, Dirienzo W. Shums A. NortonKoger B, Fudenberg HH: Defect of autologous mixed lymphocyte reac tion and interleukin-2 in aged indi viduals. Mech Ageing Dev 1985:32: 205-212.
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5 Wekslcr ME. Moody CE, Ostry RF, Casazza BA: Lymphocyte transfor mation induced by autologous cells. X. Soluble factors that generate cy totoxic T lymphocytes. J Exp Med 1980; 152:284S. 6 Stobo J, Tomasi T: Aging and the regulation of immune reactivity. J Chronic Dis 1975;28:437-440. 7 Zharhary D, Klinman N: Antigen responsiveness of the nature and generative B-ccll populations of aged mice. J Exp Med 1983:157: 1300-1328. 8 Hausman PB. Stites DP. Stobo JD: Antigen reactive T cells can be acti vated by autologous macrophages in the absence of added antigen. J Exp Med 1981;153:476-485. 9 Pope RM, McChesney L, Talal N. Fischbach M: Characterization of the defective autologous mixed lym phocyte response in rheumatoid ar thritis. Arthritis Rheum 1984;27: 1234-1244. 10 Vande Stowe R, Kunkel HG, Halper J, Wekslcr M: Autologous mixed lymphocyte culture reactions and generation of cytotoxic T cells. J Exp Med 1977;146:1809-1815. 11 Hazelton RA, Morrison JJ, Vedam R. Siskind V: Analysis of the re sponding and stimulating cells in the AMLR of patients with R.A. using limiting dilution. Clin Exp Immunol 1988;74:94-99. 12 Sakane T. Steinberg AD, Green I: Failure of autologous mixed lym phocyte reactions between T and non-T cells in patients with systemic lupus erythematosus. Proc Natl Acad Sci USA 1978;75:3464-3469. 13 Sakane T, Green I: Specificity and suppressor function of human T cells responsive to autologous non-T cells. J Immunol 1979:123:584. 14 Sakane T. Takada S: Possible in volvement of the CD4 molecules in a late activation event on CD4+ Tcell proliferation the autologous MLR. Clin Exp Immunol 1989:75: 269-274. 15 Huber C, Merkensahlater M. GattringerG. Royston I, Fink U. Braunsteiner H: Human autologous MLR is primarily specific for xenoprotein determinants adsorbed to antigenpresenting cells during rosette for mation. J Exp Med 1982:155:1222.
McGill University School of Medicine, Thorp Laboratories of Clinical Immunology, Royal Victoria Hospital, Montreal, Que., Canada
Keywords Immunology Aging AMLR Alzheimer’s
Immune Reactivity in Aging Autologous Mixed Lymphocyte Responses
Abstract The level of activity in the autologous mixed lymphocyte reac tion (AMLR) was studied in populations of young and old sub jects. AMLR activity was reduced in the older age group. A subpopulation of the older age group who had Alzheimer’s dis ease was shown to have the lowest AMLR values. Within this group, those with a history indicating a more rapid develop ment of CNS-related disability from senescence showed the weakest AMLR responses. The AMLR values correlated nei ther with sex. nutritional status nor history of infections. This impairment of a central regulating immune reaction may be a significant variable in manifestations of the aging process.
Introduction Many of the functional elements of the immune system show some decrease in activ ity with aging [1-3]. This decline is accompa nied by an increased incidence of diseases associated with impairment of immune regu latory and host defense functions [3], Because of the key importance of the immune system in the overall body economy, theories have been developed to explain the aging process through the effects of progressive immuno logic defects [4, 5]. Individual components of the functioning immune system may decline at different
This work was supported through grants from the Canadian Geriatric Society and the Lucie and Ronald Javitch Fund.
Accepted: March 21,1992
rates. Thus, an immune regulation distur bance may result when only the T-helper or T-suppressor inducer subsets are involved [6, 7], Aging patients may develop serum autoan tibodies or a monoclonal gammopathy in the absence of an underlying autoimmune disease or plasma cell dyscrasia. The elucidation of the mechanisms and the phenomena that are involved in the agerelated changes of the immune system might lead to an appreciation of possible therapeutic approaches aimed at correcting some of these, and thereby preventing the negative sequelae on the welfare of the host.
C.K. Osterland Thorp Professor of Medicine c/o Royal Victoria Hospital 687 Pine Avenue West Montreal.Que. H3A 1A1 (Canada)
© 1992 S. Kargcr AG. Basel 0304-324X/92/ 0386-030152.75/0
Downloaded by: King's College London 137.73.144.138 - 1/17/2019 2:36:41 AM
C.K. Osterland E.A. St. Louis
302
Osterland/St. Louis
Materials and Methods Patients and Subjects Three groups of patients were studied. The first consisted of young normal subjects with a mean age of 27 (range 22-36 years). The second was a population of ’normal’ aged individuals, mean age 73 (range 6774 years). These individuals exhibited a normal aging pattern and no evidence of active disease that could influence the results of laboratory studies. The third population group was hospitalized for age-related dis ability and suffered from Alzheimer’s disease (mean age 74, range 50-86 years). Blood sampling was made on a voluntary basis, with informed consent. In the case of individuals in the Alzheimer’s population, blood was acquired with family consent and was done at the same time as the patient was having venipunc ture for other diagnostic testing ordered by the attend ing physicians. The studies were approved by the Ethics Review Committee of the institution. Selection of study individuals was made with care taken to exclude any with active illnesses or those who were receiving medications that could influence the test results. The exclusion criteria used were similar to those described by Ligthart et al. [27] in the Senieur protocol. This meant that patients with infections, malignancy and malnutrition were excluded. Labora tory evidence of hypoproteincmia, renal failure, marked anemia or lymphopenia was also a basis for patient exclusion. Patients who were receiving atarac tic or antidepressive medications that may have had minor effects on in vitro immune function had the medication withdrawn 24 h prior to blood sampling. A review of the patients’ charts noted primary and secondary diagnoses. For the Alzheimer’s disease study group, a clinical judgement was made regarding two additional aspects of the patients’ disability: the first involved a global assessment of the severity of the clinical disability; the second noted the rapidity or pace with which the disability symptoms had pro gressed, from the time that symptoms were first docu mented until the time of study. A designation o f’rapid’ was assigned when the patient status had deteriorated to full disability within 6 months, and as ’slow'’ when 1 year or more elapsed between symptom onset and the occurrence of marked disability. These estimates were made independently by two observers with agreement in the classification. The patients’ nutritional status was noted objec tively and in terms of the dietary intake as indicated in the hospital chart. The frequency and nature of all doc umented infectious episodes occurring over the pre ceding 12 months were also tabulated.
AMLR in Aging
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The autologous mixed lymphocyte reac tion (AMLR) has been looked upon as having some importance in determining the func tional state of the immune system [4, 5]. The AMLR provides an in vitro means of cellular communication that stimulates the genera tion of cytokines and effector cells, in a non antigen-dependent fashion [4, 5. 8-10], In the AMLR assay, CD4+ T cells give a prolifera tive response to autologous MHC class II anti gens in the absence of known processed pep tide antigen [11-14], The presence of exoge nous antigens in the in vitro test system [15] does not explain the reactivity. While the dis play of processed specific peptide, derived from autologous or exogenous sources in the MHC class II groove is crucial to T-cell activ ity and the immune response, the AMLR reaction is predominantly dependent on the MHC class II alone. The AMLR involves the activation of greater numbers of T cells than is seen in T-cell response to a conventional peptide-MHC complex antigen. In this re gard, it is more similar to an allogeneic re sponse [5, 8. 16-22]. The activation of autoreactive T cells with specificity for self-MHC may represent a means by which self- versus non-self-discrimination and autoimmunity are regulated [ 17], The AMLR has been studied in the normal aging population as well as in many disease states [23-26], While most studies indicate a reduced activity and generation of effector cells, there has not been complete agreement. Fournier and Charriere [24] noted an increase in AMLR responses in the female subjects of their aging population study. In the current study, we carried out AMLR studies on a normal-aged population, an Alz heimer’s group and a group of young normal subjects. Reduction in AMLR was noted in the older group and was even more striking in the Alzheimer’s patients.
Rheumatoid and Antinuclear Factor Measurements Two non-organ-specific autoantibody activities, both known to occur with increased frequency in the sera of older patients, were measured. The results were subsequently correlated with the AMLR experiments. The antinuclear factors (ANA) were done by an indi rect immunofluorescence technique using human epi thelial Hep-2 cells as substrate (Quantafluor, Kallestad Laboratories Inc.), sera were screened at a 1/40 dilu tion. This technique detects serum autoantibodies spe cific for several nuclear and cytoplasmic constituents. There were no positives in the normal control group, although the test can be positive in less than 2.5% of a blood donor population. Rheumatoid factor (RF) analysis was by a standard immunochemical latex agglutination reaction (Rapi Tex RF, Behringwerke AG). Sera giving negative re sults in this assay contained no RF or had concentra tions < 20 IU/ml. Using this technique, the incidence of false positives in a normal healthy population is < 1%. As with the ANA autoantibody testing, there were no RF-positive individuals among the normal controls studied.
Results The AMLR results indicated differences among the population groups studied. The values for the aged population were lower
Table 1. AMLR results from a group of patients with Alzheimer’s disease and from two different age groups of normal controls Study group
n
Mean value ± SEM
Alzheimer’s 'Normal' aged ‘Normal’ young
24 9 11
4,876± 825 8,540 ±1,404 13,204 ±1,854
AMLR results are expressed as counts per minute 3H-thymidine incorporation minus the spearate back ground counts of B and T cells. AMLR was significantly (Mann-Whitney rank test) lower in Alzheimer’s patients than in either the normal aged (p < 0.05) or young (p < 0.01) groups. Differences in AMLR proliferative responses be tween the two normal groups failed to attain statistical significance at the p = 0.01 level.
than those of the younger, normal subjects (ta ble 1). Among the total aged population stud ied, those patients with a diagnosis of Alz heimer’s disease with related disability gave significantly lower values of 3H-thymidine in corporation than did ’controls’ in the similar age group not exhibiting Alzheimer’s demen tia or disability. Though the normal aged group had lower uptake values than the normal young group, the values did not reach statistical signifi cance (table 1). When all of the patients were grouped according to three defined age groups, there appeared to be a decrease in AMLR values that correlated with increasing age (fig. 1). Because some individual age pop ulations were poorly represented in the study, these results indicate only the trend of low ered AMLR with increasing age of these par ticular groupings. When the data were analyzed comparing male and female patients, no significant AMLR differences could be attributed to this variable (table 2).
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AMLR Heparinized blood samples were separated on a Ficoll-Hypaque (Pharmacia) gradient. Monocytes were removed by adherence to plastic dishes. T- and B-lymphocytc populations were obtained by E-rosetting the peripheral blood lymphocytes (PBL), then passing the mixture over another Ficoll-Hypaque gra dient. PBL could also be separated into T and non-T on a nylon wool column. The rosette-prepared lym phocytes and the nylon wool purified ones gave similar results. The responser T lymphocytes (1 X 105) were cultured with 1 X 105 stimulator B lymphocytes that had been inactivated with mitomycin C, 50 pg/ml (Flow Labs.). Cells were cultured in triplicate in U-bottom 96-well Linbro microtiter plates in RPMI 1640 supplemented with 10% human AB serum, 2 mM Lglutamine, 10 ntW Hepes buffer and containing 50 pg/ml gentamycin. Cultures were continued for 6 days, and then pulsed with 1 pCi 3H-thymidine for 16 h prior to harvesting and processing for counting.
n
Negative
cpm x 103
m Positive
3
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Fig. 1. AMLR proliferation (counts per minute 3Hthymidine incorporation - background counts of B and T cells) grouped according to age alone. Average proliferative responses in the individuals comprising the 60-69 and 70-79 year age categories were similar and were analyzed as a single 60-80 year age group. Although AMLR responses appeared to decrease with increasing age, the differences between groups were not statistically significant. Fig. 2. AMLR proliferation (expressed as counts per minute 3H-thymidine incorporation - background counts of B and T cells) in 23 patients with Alzheimer’s disease. Patients were subsequently grouped according to whether disease progression was judged gradual or rapid (see Materials and Methods). AMLR prolifera tive responses were lower in those patients who had experienced rapid degeneration (p < 0.05, Student’s t test). Fig. 3. AMLR proliferation (expressed as counts per minute 3H-thymidine incorporation) in 18 patients with Alzheimer’s disease. Patients were grouped ac cording to whether or not their sera tested positive for serum autoantibodies (see Materials and Methods). AMLR proliferative response were lower in the group whose sera contained detectable autoantibody activity (significant at 5% level, Mann-Whitney U test).
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cpm x 103
f~~l Gradual I I Rapid
As described in Methods, the Alzheimer’s patients were grouped according to the pace at which each progressed clinically, in terms of their symptoms of senescence and disability. Patients were assigned either to a rapid or slow progression category. Those with whom this judgement could not be made with cer tainty were excluded. Figure 2 shows that the rapid progressors had significantly lower AMLR responses than the slow progressors. The age distribution was similar in these groups as was the overall senescence disability status at the time of examination. These sub jects represented the younger population group of table 1 and figure 1. The presence of the reduced AMLR values correlates posi tively (p < 0.05) with the presence of positive tests for the non-organ-specific autoantibod ies (fig. 3).
Table 2. AML R results grouped and analyzed according to sex
Alzheimer’s
‘Normal’ aged
‘Normal’ young
M n=7
F n= 17
M n=6
F n=3
M n=2
F n=9
4,524 (1,094)
5,049 (1,092)
8.683 (1.837)
8.255 (2,594)
16.769
12.417 (2.039)
Estimates of nutritional status of the hospi talized senescent patient group and the inci dence of infections over 1 year did not show any significant correlation with AMLR values or with the patient grouping as a whole. The nursing care received by these patients was attentive, and the dietary intake and nutri tional status of even the Alzheimer’s group appeared to be excellent. The frequency of infections was surprisingly low in all patient groups.
Discussion The AMLR response is a reaction of T lymphocytes against MHC class II related an tigens on B lymphocytes [21, 28], The reac tion occurs as a result of mixing an individu al’s T and B cells together in a ratio of about 1:1, thus exposing, in vitro, the patient's T lymphocytes to an unusually high frequency of Ia-antigen-positive B lymphocytes. The reaction is quite similar to the syngeneic mixed lymphocyte culture reaction in mice [4], While some studies have indicated that exogenous foreign antigens derived during cell separation procedures or culture condi tions play a major role in the response by gen erating an in vitro immune response [ 15], this is not a complete explanation for the phenom enon [5, 8, 16-22], The AMLR stimulation
response is not as vigorous as an allogeneic response by the same cell populations, but it is markedly greater than a response to individ ual antigen [29], Regardless of the sources of any antigen or antigenic peptides that might be associated with the MHC class II mole cules in the AMLR, the patient’s T cells pro duce a polyclonal response with the genera tion of effector cells. The primary responding cell is the CD4+ T-helper cell [12, 20, 30], It is typical, how ever, to see the generation of increased num bers of activated T-helper (CD4) and T-suppressor(CD8) lymphocytes [5, 13,20, 31 ] as a result of accompanying cytokine production, especially of interleukin-2. Secondary auto crine stimulation also occurs in the cell mix tures, causing CD8 and B-cell stimulation as well. In a physiological sense, the AMLR reac tion has been considered as one that may, in an in vivo form, exert some element of im mune regulation and immune response am plification because of its action of generating increased amounts of helper, suppressor and lymphocytotoxic cell (NK) activity [32], The AMLR responses have been found to be reduced in a variety of different medical conditions, especially those that affect the im mune system through autoimmunity or lym phoma [9, 12, 17, 25, 29, 33], In systemic lupus erythematosus patients showing re duced AMLR, there appears to be an impair
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Values represent counts per minute 3H-thymidine incorporation mi nus background counts of stimulators and responders. Values in parenthe ses are standard errors.
ment of the generation of suppressor cells in the reaction [ 12], a finding considered to be of some relevance to the course of lupus itself. Results from studies on the relationship of age and senescence in the AMLR response have been somewhat controversial. Fournier and Charriere [24] reported an increase in AMLR activity in older women. Most investi gators have reported a decrease in the activity regardless of sex [23. 25, 26, 34]. If AMLR activity does become reduced with age. its proposed physiological role in immune regu lation could play an important role in the occurrence and progression of the other known defects in immune function seen with aging, such as loss of suppressor-cell function, increased autoantibody formation and re duced effector cells with poor immune sur veillance mechanisms. The studies reported here support the pre vious findings of reduced AMLR activity in the aged population, with no differences noted between the sexes. It has been pointed out [27] that immunogerontologic studies re quire careful patient selection using a proto col similar to the Senieur grouping before ageor disease-related correlations can be made. A subpopulation of the aged suffering from de bilitating Alzheimer's was found to have the lowest AMLR values. It is not clear what this finding signifies, beyond the possibility that impairment of the immune system might be one of numerous factors that contribute to the acceleration and severity of the aging process.
It was of interest that patients judged to exhibit the most rapid pace of age-related symptom progression also showed the great est reduction in their AMLR responses. One of the most important goals for human aging studies is to develop strategics for min imizing the handicaps of old age and to find better therapies for preventing geriatric dis eases and disabilities. When one finds a com mon age-related change in 'normal subjects’, this does not indicate that the change is neces sarily harmless. It may act as a cofactor to damage, coming from other predisposing fac tors. Examples of such cofactors in aging might include the effects of an elevation in blood pressure or blood glucose values. Per haps it is most important to know what changes occur as a function of normal aging. It may be possible to correlate changes in these variables in subjects identified as exhibiting minimal, moderate or advanced aging charac teristics. Most age-related alterations in the activity parameter of a biological response are likely to show only a percentage change from normal. The signals identifying such changes, therefore, may be low in a quantitative sense yet be quite significant for the individual.
Acknowledgements The authors acknowledge the assistance of Ms. Elisa Monaco and Mrs. L. Warner in the preparation of the manuscript, and the staff of the Douglas Hospi tal in obtaining patient materials for study.
I Wade A. Szewczuk M: Aging, idiotvpe shifts, and compartmentalization of the mucosal associated lym phoid system: in Kunkel H. Dixon F (eds): Advances in Immunology. New York. Academic Press, 1985. vol 36.
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2 Szewczuk M. Wade A: Cellular aging, idiotype repertoire changes and mucosal-associated lymphoid system; in Sauer HW (ed): Cellular Ageing. Monogr Dev Biol. Basel. Karger. 1984, vol 17. pp 122-141.
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3 Makinodan T. Kay MMB: Age in fluences on the immune system. Adv Immunol 1980:29:287-330. 4 Wekslcr M. Kozak R: Lymphocyte transformation induced by autolo gous cells. J Exp Med 1977:146: 1833.
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