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Monocyte function in ageing humans
Mechanisms of Ageing and Development, 16 (1981) 233-239 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
233
M O N O C Y T E F U N C T I O N IN A G E I N G H U M A N S
IAN D. GARDNER a'*, STEPHEN T. K. LIM b and JOHN W. M. LAWTON a Departments of apathology and b Surgery, University of Hong Kong (Hong K ongJ
(Received October 22, 1980; in revised form February 21, 1981)
SUMMARY Peripheral blood monocytes from hospitalised patients > 6 0 years of age and <35 years, and those from healthy normal controls <35 years, were tested for a range of functional and physiological properties, comprising chemotaxis under agarose, the ability to phagocytose and kill Candida albicans, adhesion to glass and spreading on glass. No significant difference was found between young and old groups, nor between hospitalized and non-hospitalized groups in respect of any parameter. There was some decline in phagocytosis and in spreading in a very old subgroup ('>75 years), but this was not statistically significant. This study showed that phagocytic cell function in the elderly does not decline at the same rate as the specific immune response and thus cannot directly account for the increased incidence of infection in the aged.
INTRODUCTION Advancing age is associated with an increase in the incidence of tumors [1], autoimmune diseases [2], and infections [3, 4]. Extensive studies in experimental animals and humans have shown that aspects of the specific immune response decline markedly with age, and it is thought that this many account in part for the increased incidence of these three types of disease. Resistance to infections relies heavily on the functional capability of the phagocytic cells of the body, as demonstrated by the extreme susceptibility to infections of people with phagocyte dysfunction [5]. In view of the enhanced susceptibility of the aged to infection, it is possible that the capacity of granulocytes and/or monocytes to deal with infectious organisms declines with age. Phair et al. [6] showed that polymorphonuclear leukocyte function was normal in aged subjects. In this study we examined a range of functional and physical characteristics of human monocytes in groups of young and old people. *Correspondence and reprint requests to: Dr. I. D. Gardner, Department of Pathology, University of Hong Kong, Queen Mary Hospital Compound, Hong Kong.
234 PATIENTS AND METHODS
Study population Volunteers were recruited from patients admitted for minor surgical procedures to the Department of Surgery, Queen Mary Hospital. Patients were excluded from the study if they were suffering from diabetes, hepatic dysfunction, neoplasia, collagen-vascular disease, renal disease, cardiac failure or chronic infection. Patients taking anti-inflammatory or immunosuppressive drugs were similarly excluded, as were those with acute infections. A total of 37 people older than 60 years (20 male, 17 female, mean age 70 years) and 37 between the ages of 12 and 34 years (19 male, 18 female, mean age 23 years) were the hospitalized groups. A further 35 healthy laboratory volunteers (19 male, 16 female, mean age 27 years) served as a normal control population. A number of individuals whose leukocyte count exceeded 7600 (indicating acute bacterial infection) were not included in the study. Isolation of monocytes Samples of 20 ml of venous blood were taken: 18 ml were anticoagulated with heparin 10 U/ml (preservative-free, Abbott Laboratories) and 2 ml with EDTA. Heparinized blood was diluted 1:1 in Medium 199 (M199) and a monocyteenriched population of mononuclear cells separated on a modified Ficoll-Hypaque buoyant density gradient, SG 1.077 [7]. Cytocentrifuged preparations of these cells stained with Wright's stain were used to establish differential counts. Differential and total counts of blood leukocytes were obtained by conventional haematological methods on the EDTA sample. All procedures except chemotaxis were carried out in M199 containing 20% autologous plasma removed from the top of the gradient. Chemotaxis The chemotactic responsiveness of monocytes to Zymosan-activated serum was measured by using migration under agarose gel, essentially as described by Nelson et al. [8] except that pooled normal human plasma (heparinized) was used to prepare the agarose and the cell suspension medium. This method was found to be simpler to perform, more easy to quantitate and more reproducible in our laboratory than the traditional filter assay. The net distance of migration (directed minus random) towards the chemotaxin was measured directly using a microscale fitted to a microscope eyepiece. Each result was the mean of 4 - 6 replicate assays and was expressed in micrometers. Phagocytosis and killing of Candida albicans A 5-day 30 °C culture of Candida was used to measure phagocytosis and killing by a modification of the acridine-orange staining method [9]. Cell suspensions containing 106 monocytes, 1.5 X 106 Candida and fresh frozen human plasma were spotted into wells of 4-chamber culture slides (Lab Tek), incubated for 1.5 h at 37 °C in a humidified atmosphere of 5% CO2 and washed gently with warm phosphate-buffered saline (PBS) to remove non-adherent ceils. The chamber was removed and the monolayers stained for 45
235 sec with acridine orange in Geys Balanced Salt Solution (14.4 mg/litre), rinsed in PBS and a wet mount prepared for fluorescence microscopy. Using incident ultraviolet illumination, 100 cells were examined and the total numbers of green (viable) and orange/ red (nonviable) intracellular Candida recorded. Phagocytic index was the mean number of Candida per cell; the killing index was the proportion of nonviable intracellular Candida expressed as a percentage.
Adhesion and spreading Adhesion of monocytes to glass was measured after incubation of 7.5 × 104 mononuclear cells in 0.05 ml in wells of 4-chamber tissue culture slides at 37 °C for 1.5 h in a humidified atmosphere of 5% CO2, three gentle washes with warm PBS and aspiration of the washings into a single tube. The aspirated cells were counted and a differential count made on a Wright's stained cytocentrifuge preparation. The percentage adhesion was calculated from the total monocytes plated and the number of monocytes not adhering. To measure monocyte spreading on glass the same slide was stained for 10 min with 0.1% toluidine blue in normal saline, the excess stain removed and a wet mount prepared. The longest diameter of 100 cells was measured under oil immersion using a microscale and the mean diameter at 1.5 h calculated. A second slide was similarly washed after 1.5 h, but was reincubated in medium for 24 h, after which the medium was removed, the cells stained and the mean cell diameter measured as above. A minority of cells, putting out fine cytoplasmic processes at 24 h, were excluded from the count.
RESULTS
Isolation of monocytes Blood mononuclear cell separation on a modified Ficoll-Hypaque gradient system yielded consistently high proportions and yields of monocytes. The proportion of monocytes (mean --- S.D.) in the separated cell suspension ranged from 53.6 + 10.8 (>60 years) to 53.8 + 11.9 (>75 years). Contaminating polymorphs were usually less than 10%, ranging from 6.2 -+ 3.9% (<35 years, non-hospitalized) to 7.9 _+9.6% (>75 years) and the remainder of the cells were lymphocytes. There was an occasional sample which gave an uncharacteristically high proportion of polymorphs (15-42%). These samples occurred quite randomly with respect to age, sex and time, so probably represented a technical artifact. Results from these individuals computed as a group were not significantly different from the remaining samples (data not shown) so they were included in the study.
Chemotaxis The chemotactic response of monocytes to the standard chemotaxin showed no significant variation due to age (Table I).
236 TABLE I MONOCYTE CHEMOTAXIS UNDER AGAROSE AND PHAGOCYTOSIS AND KILLING OF CANDIDA ALBICANS
Chemotaxis (l~m)
Phagocytic index
Percentage killing
1515 -+ 235 1450 ± 265 1470 +- 180
1.50 ± 0.31 1.62 +-0.40 1.72 ± 0.41
22.1 -+ 6.0 20.1 ± 5.5 20.8 +-4.7
1520 -+ 250
1.71 +_0.35
21.6 -+ 6.3
Hospitalized >75 years >60 years <35 years Non-hospitalized < 35 years
All values are expressed as mean ± S.D.
Phagocytosis and killing o f Candida albicans The mean phagocytic indices of monocytes from people under the age of 35 years were very similar in both hospitalized and non-hospitalized groups (Table I). Phagocytosis was slightly less in people > 6 0 years and less again in those > 7 5 years. The difference between those < 3 5 years and those > 7 5 years was not statistically significant. The mean percentage killing of Candida (Table I) was not significantly different in the four groups, ranging from 20.1 -+ 5.5% ( > 6 0 years) to 22.1 -+ 6.0% ( > 7 5 years). Adhesion and spreading The percentage adhesion to glass was lower in monocytes from hospitalized people than in normal controls (85.4-88.0% compared with 89.8%), but these values were not significantly different (Table III). Adhesion tended to decline with age in hospitalized people, again not reaching levels of significance. Percentage and incremental spreading in 24 h were almost identical to monocytes from normal controls and hospitalized subjects < 3 5 years (Table II). Both these parameters showed a decrease in people > 6 0 years and a further decrease in those > 7 5 years, but the decreases were not statistically significant.
DISCUSSION Ageing humans are more susceptible than the young to a wide range of infectious diseases [3, 4, 10]. Studies have been carried out in ageing humans and laboratory animals to determine the role of the immune response in this decline in resistance. The humoral immune response is depressed in aged humans [11, 12], but a more distinct decline is seen in the cell-mediated immune response. Compared with lymphocytes from the young, lymphocytes from aged humans show reduced ability to respond in culture to mitogens [6, 11, 13] and to allogeneic cells in the mixed lymphocyte reaction [13,
237 TABLE II MONOCYTE ADHESIONAND SPREADING Adhesion
(~)
Spreading Diameter (1.5 h) (tan)
Diameter (24 h} (tan)
Increase in cell diameter
Incremental increase
(%)
(urn)
Hospitalized > 75 years 85.4 -+5.0 >60 years 86.4 +-5.5 <35 years 88.0-+ 3.3
10.55 -+0.36 10.39 +-0.26 10.44+-0.33
14.02 +-0.98 14.00 -+0.95 14.20-+ 1.17
32.7 ± 8.0 34.6 -+ 8.3 36.2± 11.1
3.47 -+0.86 3.60 -+0.86 3.76_+ 1.17
Non-hospitalized <35 years 89.8 ± 2.7
10.27 -+0.16
14.03 +-0.88
36.5 -+ 8.5
3.76 -+0.85
All values are expressed as mean -+ S.D. 14]. Old people also show a reduction in magnitude and frequency of delayed-type hypersensitivity skin reactions [6, 11, 15]. It seems probable that decline in specific immune function is not the only factor involved in the increased susceptibility of the aged to infection. Observed increases in morbidity and mortality are not due to pathogens usually dealt with by the cellmediated immune response, but rather seem to be related to sites of infection such as the respiratory system [4]. Therefore the role of nonspecific physiological and cellular factors is probably important. Phagocytic cell function has been studied in aged experimental animals. Most studies in ageing mice have shown normal or enhanced macrophage function [16-19]. Gardner and Remington [20] showed normal antimicrobial levels in activated mouse peritoneal macrophages, but delayed onset of activation. In humans, studies have been restricted to polymorphonuclear leukocytes; chemiluminescence declines in very old people [21], but neutrophil turnover is increased [22] and intracellular killing capacity is not impaired [6, 23]. The monocyte studies reported here showed no statistically significant differences between old and young people in a variety of functional and physiological characteristics of monocytes. However, two properties showed a trend which could reach levels of significance if a larger number of very old people were studied. Phagocytosis was slightly reduced in people > 6 0 years and further reduced in people > 7 5 years. A similar observation was made for spreading, and this corresponds to the decline in spreading ability of peritoneal macrophages from aged mice [24]. In general, the parameters considered in this study did not show the wide spread of values observed in studies of specific immune parameters in ageing humans [11,25]. Hence, it seems that monocyte function does not decline markedly with age. There is a suggestion of a decline at extreme old age, but this is minor compared with the
238 observed reduction in the specific immune response. However, the tests performed in this study were restricted to measurements of physical and nonspecific functional characteristics of monocytes and do not involve the interaction of these cells with lymphocytes or lymphokines. Therefore, it is possible that the monocyte (macrophage) may be involved in the enhanced susceptibility of the aged to infection via its roles in induction and as an effector mechanism of the specific immune response. It has been shown that phagocytic cells from ageing mice have normal antimicrobial capacity but their function is impaired by a decline in the immunologically mediated mechanism of activation [20]. A reduction in the production of opsonising antibody or of monocyte chemotactic factor by T lymphocytes could have a similar effect in humans of reducing the effectiveness of monocytes, even though their intrinsic functional capabilities are not impaired.
ACKNOWLEDGEMENTS We thank Miss Kannie Chan and Mr. Stephen Ho for technical assistance.
REFERENCES 1 R. Doll, C. Muir and J. Waterhouse (eds.), Cancer Incidence in Five Continents, Vol 2, IUCC/ Springer-Verlag, Berlin, 1970. 2 H. T. Blumenthal and A. W. Berns, Autoimmunity in aging. In B. L. Strehler (ed.), Recent Advances in Gerontological Research, Academic Press, New York, 1964, pp. 289-342. 3 J. L. Gladstone and R. Recco, Host factors and infectious diseases in the elderly. Med. Clin. North Am., 60 (1976) 1225-1240. 4 I. D. Gardner, The effect of aging on susceptibility to infection. Rev. Infect. Dis., 2 (1980) 801-810. 5 J. A. Bellanti and D. H. Dayton (eds.), The Phagocytic Cell in HostResistance, Raven Press, New York, 1975, pp. 173-293. 6 J. P. Phair, C. A. Kauffman, A. Bjornson, J. Gallagher, L. Adams and E. V. Hess, ttost defenses in the aged: evaluation of components of the inflammatory and immune responses. J. lnfect. Dis., 138 (1978) 67-73. 7 J. W. M. Lawton and 1. D. Gardner, Monocyte function in normal adults: simplified methods of separation and testing. Scand. J. Haematol., in press. 8 D. L. Nelson, P. G. Quie and R. L. Simmons, Chemotaxis under agarose: A new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol., 115 (1975) 1650-1656. 9 D. L. Smith and F. Rommel, A rapid micro method for the simultaneous determination of phagocytic-microbicidal activity of human peripheral, blood leukocytes in vitro. J. lmmunol. Methods, 1 7 (1977) 241-247. 10 D. Perla and J. Marmorston, Natural resistance in old age. In Natural Resistance and Clinical Medicine, Little, Brown, Boston, 1941, pp. 363-382. 11 I. C. Roberts-Thomson, S. Whittingham, U. Youngchaiyud and 1. R. Mackay, Ageing, immune response, and mortality. Lancet, ii (1974) 368-370. 12 C. H. L. Howells, C. K. Vesselinova-Jenkins, A. D. Evans and J. James, Influenza vaccination and mortality from bronchopneumonia in the elderly. Lancet, i (1975) 381-383. 13 M. E. Weksler and T. H. Hutteroth, Impaired lymphocyte function in aged humans. J. Clin. Invest., 53 (1974) 99-104.
239 14 S. Kishimoto, S. Tomino, K. Inomata, S. Kotegawa, T. Saito, M. Kuroki, II. Mitsuya and S. Hisamitsu, Age-related changes in the subsets and functions of human T lymphocytes. J. lmmunoL, 121 (1978) 1773-1780. 15 J. P. Girard, M. Paychere, M. Cuevas and B. Fernandes, Cell-mediated immunity in an ageing population. Clin. Exp. Immunol., 27 (1977) 85-91. 16 W. Cantrell and E. E. Elko, Effect of age on phagocytosis of carbon in the rat. Exp. Parasitol., 34 (1973) 337-343. 17 B. N. Jaroslow and J. W. Larrick, Clearance of foreign red cells from the blood of aging mice. Mech. Ageing Dev., 2 (1973) 23-32. 18 M. Bar-Eli and R. Galllly, Age-dependent macrophage functions in New Zealand black mice. Cell Immunol., 45 (1979) 309-317. 19 R. E. Callard, Immune function in aged mice. III. Role of macrophages and effect of 2-mercaptoethanol in the response of spleen cells from old mice to phytohemagglutinin, lipopolysaccharide and allogeneic cells. Eur. J. Immunol., 8 (1978) 697-705. 20 I. D. Gardner and J. S. Remington, Aging and the immune response. II. Lymphocyte responsiveness and macrophage activation in Toxoplasma gondii-infected mice. J. lmmunol., 120 (1978) 944-949. 21 D. E. van Epps, J. S. Goodwin and S. Murphy, Age-dependent variations in polymorphonuclear leukocyte chemiluminescence. Infect. Immun., 22 (1978) 57-61. 22 P. Resnitzky, M. Touma and D. Danon, Neutrophillc turnover rate in human age groups evaluated by serum lysozyme activity. Gerontology, 24 (1978) 111-116. 23 J. Palmblad and A. Haak, Ageing does not change blood granulocyte bactericidal capacity and levels of complement factors 3 and 4. Gerontology, 24 (1978) 381-385. 24 D. R. Johnson, G. Fernandes and S. D. Douglas, Age related decline in cytoplasmic spreading of mouse peritoneal macrophages. Dev. Comp. Immunol., 2 (1978) 347-354. 25 P. Emmerllng, H. Hof and H. Finger, Age-related defense against infection with intracellular pathogens. Gerontology, 25 (1979) 327-336.
233
M O N O C Y T E F U N C T I O N IN A G E I N G H U M A N S
IAN D. GARDNER a'*, STEPHEN T. K. LIM b and JOHN W. M. LAWTON a Departments of apathology and b Surgery, University of Hong Kong (Hong K ongJ
(Received October 22, 1980; in revised form February 21, 1981)
SUMMARY Peripheral blood monocytes from hospitalised patients > 6 0 years of age and <35 years, and those from healthy normal controls <35 years, were tested for a range of functional and physiological properties, comprising chemotaxis under agarose, the ability to phagocytose and kill Candida albicans, adhesion to glass and spreading on glass. No significant difference was found between young and old groups, nor between hospitalized and non-hospitalized groups in respect of any parameter. There was some decline in phagocytosis and in spreading in a very old subgroup ('>75 years), but this was not statistically significant. This study showed that phagocytic cell function in the elderly does not decline at the same rate as the specific immune response and thus cannot directly account for the increased incidence of infection in the aged.
INTRODUCTION Advancing age is associated with an increase in the incidence of tumors [1], autoimmune diseases [2], and infections [3, 4]. Extensive studies in experimental animals and humans have shown that aspects of the specific immune response decline markedly with age, and it is thought that this many account in part for the increased incidence of these three types of disease. Resistance to infections relies heavily on the functional capability of the phagocytic cells of the body, as demonstrated by the extreme susceptibility to infections of people with phagocyte dysfunction [5]. In view of the enhanced susceptibility of the aged to infection, it is possible that the capacity of granulocytes and/or monocytes to deal with infectious organisms declines with age. Phair et al. [6] showed that polymorphonuclear leukocyte function was normal in aged subjects. In this study we examined a range of functional and physical characteristics of human monocytes in groups of young and old people. *Correspondence and reprint requests to: Dr. I. D. Gardner, Department of Pathology, University of Hong Kong, Queen Mary Hospital Compound, Hong Kong.
234 PATIENTS AND METHODS
Study population Volunteers were recruited from patients admitted for minor surgical procedures to the Department of Surgery, Queen Mary Hospital. Patients were excluded from the study if they were suffering from diabetes, hepatic dysfunction, neoplasia, collagen-vascular disease, renal disease, cardiac failure or chronic infection. Patients taking anti-inflammatory or immunosuppressive drugs were similarly excluded, as were those with acute infections. A total of 37 people older than 60 years (20 male, 17 female, mean age 70 years) and 37 between the ages of 12 and 34 years (19 male, 18 female, mean age 23 years) were the hospitalized groups. A further 35 healthy laboratory volunteers (19 male, 16 female, mean age 27 years) served as a normal control population. A number of individuals whose leukocyte count exceeded 7600 (indicating acute bacterial infection) were not included in the study. Isolation of monocytes Samples of 20 ml of venous blood were taken: 18 ml were anticoagulated with heparin 10 U/ml (preservative-free, Abbott Laboratories) and 2 ml with EDTA. Heparinized blood was diluted 1:1 in Medium 199 (M199) and a monocyteenriched population of mononuclear cells separated on a modified Ficoll-Hypaque buoyant density gradient, SG 1.077 [7]. Cytocentrifuged preparations of these cells stained with Wright's stain were used to establish differential counts. Differential and total counts of blood leukocytes were obtained by conventional haematological methods on the EDTA sample. All procedures except chemotaxis were carried out in M199 containing 20% autologous plasma removed from the top of the gradient. Chemotaxis The chemotactic responsiveness of monocytes to Zymosan-activated serum was measured by using migration under agarose gel, essentially as described by Nelson et al. [8] except that pooled normal human plasma (heparinized) was used to prepare the agarose and the cell suspension medium. This method was found to be simpler to perform, more easy to quantitate and more reproducible in our laboratory than the traditional filter assay. The net distance of migration (directed minus random) towards the chemotaxin was measured directly using a microscale fitted to a microscope eyepiece. Each result was the mean of 4 - 6 replicate assays and was expressed in micrometers. Phagocytosis and killing of Candida albicans A 5-day 30 °C culture of Candida was used to measure phagocytosis and killing by a modification of the acridine-orange staining method [9]. Cell suspensions containing 106 monocytes, 1.5 X 106 Candida and fresh frozen human plasma were spotted into wells of 4-chamber culture slides (Lab Tek), incubated for 1.5 h at 37 °C in a humidified atmosphere of 5% CO2 and washed gently with warm phosphate-buffered saline (PBS) to remove non-adherent ceils. The chamber was removed and the monolayers stained for 45
235 sec with acridine orange in Geys Balanced Salt Solution (14.4 mg/litre), rinsed in PBS and a wet mount prepared for fluorescence microscopy. Using incident ultraviolet illumination, 100 cells were examined and the total numbers of green (viable) and orange/ red (nonviable) intracellular Candida recorded. Phagocytic index was the mean number of Candida per cell; the killing index was the proportion of nonviable intracellular Candida expressed as a percentage.
Adhesion and spreading Adhesion of monocytes to glass was measured after incubation of 7.5 × 104 mononuclear cells in 0.05 ml in wells of 4-chamber tissue culture slides at 37 °C for 1.5 h in a humidified atmosphere of 5% CO2, three gentle washes with warm PBS and aspiration of the washings into a single tube. The aspirated cells were counted and a differential count made on a Wright's stained cytocentrifuge preparation. The percentage adhesion was calculated from the total monocytes plated and the number of monocytes not adhering. To measure monocyte spreading on glass the same slide was stained for 10 min with 0.1% toluidine blue in normal saline, the excess stain removed and a wet mount prepared. The longest diameter of 100 cells was measured under oil immersion using a microscale and the mean diameter at 1.5 h calculated. A second slide was similarly washed after 1.5 h, but was reincubated in medium for 24 h, after which the medium was removed, the cells stained and the mean cell diameter measured as above. A minority of cells, putting out fine cytoplasmic processes at 24 h, were excluded from the count.
RESULTS
Isolation of monocytes Blood mononuclear cell separation on a modified Ficoll-Hypaque gradient system yielded consistently high proportions and yields of monocytes. The proportion of monocytes (mean --- S.D.) in the separated cell suspension ranged from 53.6 + 10.8 (>60 years) to 53.8 + 11.9 (>75 years). Contaminating polymorphs were usually less than 10%, ranging from 6.2 -+ 3.9% (<35 years, non-hospitalized) to 7.9 _+9.6% (>75 years) and the remainder of the cells were lymphocytes. There was an occasional sample which gave an uncharacteristically high proportion of polymorphs (15-42%). These samples occurred quite randomly with respect to age, sex and time, so probably represented a technical artifact. Results from these individuals computed as a group were not significantly different from the remaining samples (data not shown) so they were included in the study.
Chemotaxis The chemotactic response of monocytes to the standard chemotaxin showed no significant variation due to age (Table I).
236 TABLE I MONOCYTE CHEMOTAXIS UNDER AGAROSE AND PHAGOCYTOSIS AND KILLING OF CANDIDA ALBICANS
Chemotaxis (l~m)
Phagocytic index
Percentage killing
1515 -+ 235 1450 ± 265 1470 +- 180
1.50 ± 0.31 1.62 +-0.40 1.72 ± 0.41
22.1 -+ 6.0 20.1 ± 5.5 20.8 +-4.7
1520 -+ 250
1.71 +_0.35
21.6 -+ 6.3
Hospitalized >75 years >60 years <35 years Non-hospitalized < 35 years
All values are expressed as mean ± S.D.
Phagocytosis and killing o f Candida albicans The mean phagocytic indices of monocytes from people under the age of 35 years were very similar in both hospitalized and non-hospitalized groups (Table I). Phagocytosis was slightly less in people > 6 0 years and less again in those > 7 5 years. The difference between those < 3 5 years and those > 7 5 years was not statistically significant. The mean percentage killing of Candida (Table I) was not significantly different in the four groups, ranging from 20.1 -+ 5.5% ( > 6 0 years) to 22.1 -+ 6.0% ( > 7 5 years). Adhesion and spreading The percentage adhesion to glass was lower in monocytes from hospitalized people than in normal controls (85.4-88.0% compared with 89.8%), but these values were not significantly different (Table III). Adhesion tended to decline with age in hospitalized people, again not reaching levels of significance. Percentage and incremental spreading in 24 h were almost identical to monocytes from normal controls and hospitalized subjects < 3 5 years (Table II). Both these parameters showed a decrease in people > 6 0 years and a further decrease in those > 7 5 years, but the decreases were not statistically significant.
DISCUSSION Ageing humans are more susceptible than the young to a wide range of infectious diseases [3, 4, 10]. Studies have been carried out in ageing humans and laboratory animals to determine the role of the immune response in this decline in resistance. The humoral immune response is depressed in aged humans [11, 12], but a more distinct decline is seen in the cell-mediated immune response. Compared with lymphocytes from the young, lymphocytes from aged humans show reduced ability to respond in culture to mitogens [6, 11, 13] and to allogeneic cells in the mixed lymphocyte reaction [13,
237 TABLE II MONOCYTE ADHESIONAND SPREADING Adhesion
(~)
Spreading Diameter (1.5 h) (tan)
Diameter (24 h} (tan)
Increase in cell diameter
Incremental increase
(%)
(urn)
Hospitalized > 75 years 85.4 -+5.0 >60 years 86.4 +-5.5 <35 years 88.0-+ 3.3
10.55 -+0.36 10.39 +-0.26 10.44+-0.33
14.02 +-0.98 14.00 -+0.95 14.20-+ 1.17
32.7 ± 8.0 34.6 -+ 8.3 36.2± 11.1
3.47 -+0.86 3.60 -+0.86 3.76_+ 1.17
Non-hospitalized <35 years 89.8 ± 2.7
10.27 -+0.16
14.03 +-0.88
36.5 -+ 8.5
3.76 -+0.85
All values are expressed as mean -+ S.D. 14]. Old people also show a reduction in magnitude and frequency of delayed-type hypersensitivity skin reactions [6, 11, 15]. It seems probable that decline in specific immune function is not the only factor involved in the increased susceptibility of the aged to infection. Observed increases in morbidity and mortality are not due to pathogens usually dealt with by the cellmediated immune response, but rather seem to be related to sites of infection such as the respiratory system [4]. Therefore the role of nonspecific physiological and cellular factors is probably important. Phagocytic cell function has been studied in aged experimental animals. Most studies in ageing mice have shown normal or enhanced macrophage function [16-19]. Gardner and Remington [20] showed normal antimicrobial levels in activated mouse peritoneal macrophages, but delayed onset of activation. In humans, studies have been restricted to polymorphonuclear leukocytes; chemiluminescence declines in very old people [21], but neutrophil turnover is increased [22] and intracellular killing capacity is not impaired [6, 23]. The monocyte studies reported here showed no statistically significant differences between old and young people in a variety of functional and physiological characteristics of monocytes. However, two properties showed a trend which could reach levels of significance if a larger number of very old people were studied. Phagocytosis was slightly reduced in people > 6 0 years and further reduced in people > 7 5 years. A similar observation was made for spreading, and this corresponds to the decline in spreading ability of peritoneal macrophages from aged mice [24]. In general, the parameters considered in this study did not show the wide spread of values observed in studies of specific immune parameters in ageing humans [11,25]. Hence, it seems that monocyte function does not decline markedly with age. There is a suggestion of a decline at extreme old age, but this is minor compared with the
238 observed reduction in the specific immune response. However, the tests performed in this study were restricted to measurements of physical and nonspecific functional characteristics of monocytes and do not involve the interaction of these cells with lymphocytes or lymphokines. Therefore, it is possible that the monocyte (macrophage) may be involved in the enhanced susceptibility of the aged to infection via its roles in induction and as an effector mechanism of the specific immune response. It has been shown that phagocytic cells from ageing mice have normal antimicrobial capacity but their function is impaired by a decline in the immunologically mediated mechanism of activation [20]. A reduction in the production of opsonising antibody or of monocyte chemotactic factor by T lymphocytes could have a similar effect in humans of reducing the effectiveness of monocytes, even though their intrinsic functional capabilities are not impaired.
ACKNOWLEDGEMENTS We thank Miss Kannie Chan and Mr. Stephen Ho for technical assistance.
REFERENCES 1 R. Doll, C. Muir and J. Waterhouse (eds.), Cancer Incidence in Five Continents, Vol 2, IUCC/ Springer-Verlag, Berlin, 1970. 2 H. T. Blumenthal and A. W. Berns, Autoimmunity in aging. In B. L. Strehler (ed.), Recent Advances in Gerontological Research, Academic Press, New York, 1964, pp. 289-342. 3 J. L. Gladstone and R. Recco, Host factors and infectious diseases in the elderly. Med. Clin. North Am., 60 (1976) 1225-1240. 4 I. D. Gardner, The effect of aging on susceptibility to infection. Rev. Infect. Dis., 2 (1980) 801-810. 5 J. A. Bellanti and D. H. Dayton (eds.), The Phagocytic Cell in HostResistance, Raven Press, New York, 1975, pp. 173-293. 6 J. P. Phair, C. A. Kauffman, A. Bjornson, J. Gallagher, L. Adams and E. V. Hess, ttost defenses in the aged: evaluation of components of the inflammatory and immune responses. J. lnfect. Dis., 138 (1978) 67-73. 7 J. W. M. Lawton and 1. D. Gardner, Monocyte function in normal adults: simplified methods of separation and testing. Scand. J. Haematol., in press. 8 D. L. Nelson, P. G. Quie and R. L. Simmons, Chemotaxis under agarose: A new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol., 115 (1975) 1650-1656. 9 D. L. Smith and F. Rommel, A rapid micro method for the simultaneous determination of phagocytic-microbicidal activity of human peripheral, blood leukocytes in vitro. J. lmmunol. Methods, 1 7 (1977) 241-247. 10 D. Perla and J. Marmorston, Natural resistance in old age. In Natural Resistance and Clinical Medicine, Little, Brown, Boston, 1941, pp. 363-382. 11 I. C. Roberts-Thomson, S. Whittingham, U. Youngchaiyud and 1. R. Mackay, Ageing, immune response, and mortality. Lancet, ii (1974) 368-370. 12 C. H. L. Howells, C. K. Vesselinova-Jenkins, A. D. Evans and J. James, Influenza vaccination and mortality from bronchopneumonia in the elderly. Lancet, i (1975) 381-383. 13 M. E. Weksler and T. H. Hutteroth, Impaired lymphocyte function in aged humans. J. Clin. Invest., 53 (1974) 99-104.
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