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1 Key words: elderly patients, neutrophil, chemiluminescence, LPS, TNF-a
2
3 Table 1 Whole blood chemiluminescence Table 2 Neutrophil chemiluminescence Fig. 1 The average time course for non-opsonized zymosan-induced chemiluminescence (CL) of whole blood (a) and neutrophils (b) from healthy adults (-) and the elderly(---). Differences between two groups were significant (a: p= , b: p=0.0204). cpm; counts per minutes
4 Fig. 2 The integrated CL of whole blood induced by non-opsonized zymosan and various microorganisms in the elderly patients with active bacterial infection. The value of integrated CL was plotted. The open circles and the vertical lines represent means and SE, respectively. The range represented by oblique lines shows mean } SD in the elderly without bacterial infection. Differences between two groups were significant (p<0.0001). Fig. 3 The integrated CL of neutrophils induced by non-opsonized zymosan and lumispheres in the elderly with active bacterial infection. The value of integrated CL was plotted. The open circles and the vertical lines represent means and SE, respectively. The range represented by oblique lines shows mean }SD in the elderly without bacterial infection. Differences between two groups were significant (a; p < , b; p= ).
5 Fig. 4 Integrated CL of whole blood stimulated with non-opsonized zymosan in 9 elderly patients with bacterial infection tested during period of active bacterial infection and convalescence. Open circles and the vertical lines represent means and SE, respectively. Differences between two phases are significant (p=0.014). Fig. 5 Priming effects of lipopolysacchalide (LPS)(a) and tumor necrosis factor-alpha (TNF-a) (b) on whole blood CL stimulated with non-opsonized zymosan and P. aeruginosa. The CL-index of each person was plotted. The open circles and the vertical lines represent means and SD, respectively. CL-index was determined as described in the text. Differences between two groups were significant (p=0.0001).
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7 1) Yoshikawa, T. T. & Norman, D. C.: Epidemiology of infectious diseases. In Aging and Clinical Practice: Infectious Diseases.(Yo- T. T. & Norman, D. C., eds) p.3-7, shikawa, Igaku-shoin, New York, Tokyo, ) Yoshikawa, T.T. & Norman, D.C.: Predisposing factors to infection. In Aging and clinical practice: Infectious Diseases. (Yoshikawa, T. T. & Norman, D. C., eds) p.8-19, Igaku-shoin, New York, Tokyo, ) Corberand, J. X., Laharrague,P. F. & Fillola, G.: Neutrophils of healthy aged humans are normal. Mech Ageing Dev., 36: 57-63, ) MacGregor, R. R. & Shalit, M.: Neutrophil function in healthy elderly subjects. J Gerontol, 45: M55-60, ) Aoki, M., Ono, Y. & Kunii, O.: Effect of interferon-y on human neutrophil chemiluninescence. Chemotherapy (Tokyo), 40: , ) Ono, Y., Kunii, O., Kobayashi, K. & Kanegasaki, S.: Evaluation of opsonophagocytic dysfunctions in 'severely burned patients by luminol-dependent chemiluminesence. Microbiol Immunol, 37: , ) Aoki, M., Ono, Y., Kunii, O. & Gaoldstein, E.: Effect of newer quinolones on the extra- and intra-cellular chemiluminescence response of human polymorphonuclear leukocytes. J. Antimicrob. Chemother,in press. 11) Ono, Y.: Chemiluminescence response of human phagocytes in septic patients: Priming effects by endotoxin and inflammatory cytokines. Chemotherapy (Tokyo), 42: , ) Emanuelli, G., Lanzio, M., Anfossi, T., Romano, S., Anffossi. G. & Calcamuggi, G.: Influence of age on polymorphonuclear leukocytes in vitro: phagocytic activity in healthy human subjects. Gerontology, 32: , 1986.
8 14) Ewetz, L., Palmblad, J. & Thore,A.: The relationship between luminol chemiluminescence and killing of Staphylococcus aureus by neutrophil granulocytes. Ann. Hematol., 43: , ) Steele, R. W.: Clinical applications of chemiluminescence of granulocytes. Clin. Infect. Dis., 13: , ) Lipschitz, D. A., Udupa, K. B., Indelicato, S.R. & Das, M.: Effect of age on second messenger generation in neutrophils. Blood, 78: , ) Uchida, T., Kanno, T. & Hosaka, S.: Direct measurement of phagosomal reactive oxygen by luminol-binding microspheres. J. Immunol. Methods, 77: 55-61, ) Barbour, A.G., Allred, C. D., Solberg,C. O. & Hill, H.R.: Chemiluminescence by polymorphonuclear leukocytes from patients with active bacterial infection.j. Infect. Dis., 141: 14 26, ) Seres,I., Csongor, J., Mohdcsi, A., Leovey, A. & Rift, T.: Age-dependent alterations of human recombinant GM-CSF effects on human granulocytes. Mech Ageing Dev., 71: , ) Chandra, R.K.: Nutrition, immunity, and 34) Bortolussi, R., Howlett, S., Rajaraman, K. & infection: Present knowlege and future directions. Lancet, 26: , newborn cord blood-derived polymorphonu- Halperin, S.: Deficient priming activity of clear neutrophilic granulocytes with lipopolysaccaharide and tumor necrosis factor- 22) Sakamoto, M., Ishii, S., Nishioka, K. & Shimada, K.: Complement response after experimental bacterial infection in various nutritional states. Immunology, 38: , ) Chandra, R. K.: Nutrition is an important determinant of immunity in old age. Prog. Clin. Biol. Res., 326: , ) Lipschitz, D. A. & Udupa, K. B.: Influence of aging and protein deficiency on neutrophil function. J. Gerontol., 41: , ) Riley-Roberts,M. L., Evans, P. M., Turner,R. J. & Merry, B. J.: Failure of dietary restriction to 27) Solomkin, J. S., Bauman, M. P., Nelson, R. D. & Simmons, R. L.: Neutrophils dysfunction during the course of intra-abdominal infection. Ann. Surg., 194: 9-17, ) Wilson, M. E.: Effects of bacterial endotoxins on neutrophil function. Clin. Infect. Dis., 7: ) Steinbeck, M. L. & Roth, J. A.: Neutrophil activation by recombinant cytokines. Clin. Infect. Dis., 11: , ) Ohyatsu, I., Ono, Y., Aoki, M. & Kunii, O.: Effect of recombinant granulocyte colonystimulating factor on the chemiluminescence response of human polymorphonuclear leukocytes. Chemotherapy (Tokyo), 41: , phenylalanine. Pediatr. Res., 34: , ) Yasui, K., Komiyama, A., Molski, T. F. P. & Sha'afi, R. I.: Pentoxifylline and CD14 antibody additively inhibit priming of polymorphonuclear leukocytes for enhanced release of superoxide by lipopolysaccharide: Possible mechanism of these actions. Infect. Immun., 62: , ) Solomkin, J. S., Tindal, C. J., Cave, C. M. & Zemlan, F.: Regulation of TNF-a receptor expression on human neutrophils by various cell activating factors. J. Surg. Res., 56: 261 enhance cytolytic and chemiluminesent activity of peritoneal exudate cells from ageing rats. 37) Rock, G. A. W., Taverne, J. & Playfair, J. H. L.: Mech Ageing Dev., 61: , Evaluation of TNF as antiviral, antibacterial and antiparasitic agent. Biotherapy, 3: 167
9 Study of Neutrophil Dysfunctions in the Elderly Using a Chemiluminescence Method Masumi AOKI Second Department of Internal Medicine, Teikyo University School of Medicine To evaluate the cause of the vulnerability to infections in the elderly, the ability of neutrophil to generate reactive oxygen species was assessed by a luminol-dependent chemiluminescence (CL) assay after stimulation with non-opsonized zymosan, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans and lumispheres in elderly patients aged 70 to 93 years. The integrated CL for 20 minutes of whole blood and neutrophils induced by zymosan in the elderly was significantly lower than that in healthy young adults, and the integrated CL of neutrophils induced by lumispheres was also significantly lower in the elderly aged 80 years and over. When bacterial infection occurred in the elderly, the levels of CL were elevated and decreased in the convalescence. This response is proper for host-defense mechanism against infection. However, whole blood CL response was not fully activated in any patients of the elderly during bacterial infection. In these cases lower white blood cell counts, lower neutrophil counts, or the decreased level of the serum total protein, albumin, total cholesterol or cholinesterase were observed. Relationship between malnutrition and the ability of neutrophil to generate reactive oxygen species was suggested. Furthermore, I evaluated the priming effect of lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-a') on whole blood CL. The CI, responses stimulated with non-opsonized zymosan or P. aeruginosa were enhanced by pretreatment with TNF- a and LPS in healthy young adults. On the other hand, no significant priming effect was observed when blood from elderly patients were incubated with each primer. These findings suggest that the impairment in the generation of reactive oxygen species of the neutrophils and the decrease in reactivity to LPS and TNF-a that activate neutrophils at the site of infection and potentiate host defense against invading bacteria, may contribute to susceptibility to infection in the elderly.
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