The specific influence of malnutrition on the pathophysiologic changes induced by chronic alcoholism is controversial. In an attempt to determine and demarcate the effects of protein malnutrition from those produced by alcoholism and to evaluate the precise effect of alcohol per se on cytochemical and ultrastructural properties of rat polymorphonuclear neutrophil (PMN) granules, we investigated the influence of chronic protein malnutrition or chronic alcoholism alone and in combination, in rats. After a 4 month experimental period various PMN properties, such as cytochemical, morphometrical and ultrastructural, as well as neutrophil functions were studied. It was found that the degree of damage of PMNs induced either by ethanol or protein malnutrition alone was similar whereas their combination led to worsening of all markers of PMN functional ability. Ultrastructural changes of neutrophil granules including reduction, redistribution and atypical accumulation as well as appearance of autophagic vacuoles, confirmed their alteration which was emphasised by the additive pathophysiological interaction of alcoholism and chronic hypoprotein malnutrition.
Key words Alcoholism - polymorphonuclear neutrophil - protein malnutrition - ultrastructural changes
The neutrophil functions by mobilizing four essentially different types of intracellular granules and vesicles, some crucial for interaction with the endothelium and migration, others for phagocytosis and killing of microorganisms1. In addition, the production of cytokines (TNF-alpha, IL-lI, IL-1Beta, IL-1rA, IL-8 and TGF-1Beta) by polymorphonuclear neutrophils (PMNs) is a newly recognized function2.
The consumption of ethanol in large quantities has been associated with various haematologic and immunologic abnormalities3-7. Though numerous workers have commented on the toxic effect of alcohol on PMN function, their findings are inconsistent and to some extent contradictory8-12. On the other hand, protein malnutrition may be reflected by many metabolic parameters in PMNs which are associated with a large number of enzymes and functional disturbances13-17. The demarcation of malnutrition effects from those produced by alcoholism is not very clear. In some studies, the degree of damage induced by either alcoholism or malnutrition alone was found to be similar and in others the cellular damage induced by dietary deficiencies was greater than that induced by ethanol4,18-22.
In an attempt to demarcate the effects of protein malnutrition from those produced by alcohol and to determine the precise effect of alcohol per se on the cytochemical and ultrastructural properties of peripheral blood PMNs granules, we studied in rats, the effect of (i) chronic protein malnutrition; (ii) chronic alcoholism; and (iii) chronic alcoholism combined with protein malnutrition.
Material & Methods
Animals and experimental design: Two month old male Wistar rats (weight 229-249g) were devided into four groups of 15 animals each. Group I was fed on a nutritionally adequate diet without ethanol; group II on protein-deficient diet without ethanol; group III on nutritionally adequate diet with ethanol; and group IV on a protein-deficient diet with ethanol as described earlier12,16. The experiment lasted for four months. The ethanol groups (i.e., III and IV) were given free access to an aqueous solution of 24 per cent sucrose (w/w)-32 per cent ethanol (v/v) and pelleted food. The total energy intake of rats in groups I and II was the same as that of rats of the alcohol groups. The pair-feeding process was checked every 2 days, so that the mean amount of daily energy intake was nearly identical in the four groups.
Blood sampling: Blood samples for determination of blood alcohol level (BAL), white blood cell (WBC) count, leukocyte chemotaxis and phagocytic ability as well as for making smears for cytochemical investigation were taken from the tail vein. For preparing buffy coat specimens for ultrastructural examination, blood was drawn after cardiac puncture.
BAL was determined by the enzymatic method (Sigma diagnostics, kit No. 322-UV, Sigma Diagnostic, Sigma, USA).
WBC counts were made in a haematocytometer after dilution with Turk's solution. Peripheral blood leukocyte differential counts were made on 100 counted cells on blood smears.
Cytochemical and immunocytochemical investigations of neutrophils: The kits for naphthol AS-D chloroacetate esterase (NASD), neutrophil acid phosphatase (NAcP) and p-glucuronidase ([-Glu) cytochemical staining were obtained from the Sigma Chemical Co., USA (Sigma diagnostics, kit Nos. 180, 90 and 386, respectively). A PAP technique was used for identification of lysozyme (Dako PAP Kit, Denmark). The cationic bactericidal protein, myeloperoxidase (MPO), neutrophil alkaline phosphatase (NAP), periodic acid-Schiff (PAS) and Sudan black B cytochemical reactions were determined using standard procedures as recommended elsewhere23. The intensity of the cytochemical staining was graded semiquantitatively on the scale of If to 4+ per 300 neutrophils, and total scores were calculated. Electron microscopy (EM): For routine electron microscopic examination, buffy coat specimens were prepared as described previously24.
Cytochemical staining for ultracytochemical presentation of leukocyte MPO was performed essentially as described by Graham and Karnovski25. Ultrathin sections were contrasted with lead citrate before examination in an Opton 109 electron microscope (Opton, Germany).
Ultrastructural morphometry: Prints were made from negatives of nucleated neutrophils (7,000 x-original magnification). About 3-5 prints were analyzed from each animal. For stereological analysis (excluding granular radius and relative ratio of azurophilic to specific granules) test micrographs were covered with a transparent lattice point-counting grid using the method of Weibel et al26. The mean granular radius was ensured using a Kotron MOP-AMO-3 semiautomatic analyzer (Carl Zeiss, Germany). The relative ratio of azurophilic to specific granules was obtained from micrographs prepared from cytological samples in which myeloperoxidase enzyme activity was used as a marker of azurophilic granules.
Neutrophil function test:
Chemotaxic assay (spontaneous migration and chemotaxic response) - The chemotactic activity of PMNs was studied under agarose27.
Phagocytosis assay - A quantitative assessment of phagocytosis by PMNs was measured by standard method28 as described previously17. Briefly, 200 (mu)l of citrated venous blood and 100 (mu)l of one-day broth cultures of Escherichia coli O^sub 25^ (containing 2x109 bacteria) were added to round-bottomed glass tubes. The tubes were shaken for 15 min and then incubated for 20 min at 37degC. After incubation, the mixture of the blood with bacteria was used for preparing the blood smears. From these blood smears, the phagocytic activity was From these blood smears, the phagocytic activity was estimated on the basis of the following parameters: phagocytosis cell percentage (number of active, phagocytic cells per total neutrophils), Hamburger's phagocytosis index (mean number of phagocytosed microorganisms per neutrophil), Rait's phagocytosis number (mean number of phagocytosed microorganism per active neutfophil) and absolute index of ingestion (ratio between the absolute neutrophil number per mm3 and Hamburger's phagocytosis index).
Reduction of nitroblue tetrazolium (NBT) - NBT reduction was determined using the Sigma kit (Sigma diagnostics, kit No. 840-W, Sigma, USA).
Statistical analysis: All results were expressed as means +/- SE and were analyzed by two-way analysis of variance (ANOVA) using 2x2 classification (ethanol, no ethanol versus low-protein, regular protein). When indicated by the analysis of variance, individual means were compared using the Student-Newman-Keuls post hoc test.
Results
There was no significant difference among the 4 groups of animals regarding daily energy intake. However, while groups I and III had an adequate protein intake (about 25% of daily energy intake, i.e., about 6.0 g/kg/day), groups II and IV had a deficient protein intake (about 6% of daily energy intake, i.e., 1.5 g/kg/ day). All the experimental groups consumed identical amounts of fat. Ethanol contributed 23 per cent of the total energy intake in the alcohol alone group (group III) and 15 per cent in the group with both chronic alcoholism and protein malnutrition (group IV). The mean daily amount of alcohol consumed was 8.420.36 and 6.270.32 g/kg body weight for groups III and IV respectively. All diets contained adequate amounts of essential trace elements and vitamins. The rats fed on a combination of alcohol and protein deficient diet (group IV) had a lower weight at the end of experimental period compared to the other groups (Table I).
The absolute leukocyte count was not significantly different among the 4 groups (data not shown) but the rats fed on ethanol alone (group III) showed a significant increase (P
MPO enzyme score was unaffected under experimental conditions. The semiquantitative cytochemical properties which were significantly (P
A summary of the quantitative analysis of ultrastructural morphometry of neutrophil granules is shown in Table III. The PMNs of control rat (group I) with characteristic ultrastructural properties are shown in Figs 2a and b. It was found that both protein deficiency as well as ethanol feeding reduced the total number of cytoplasmic granules per cell profile (Figs 3 and 4) and that their effects were additive. Also, reduction of the volume density of granules as well as mean granular diameter was observed in PMNs from both protein-deficient animals (group II) and animals fed on ethanol in combination with a protein-deficient diet (group IV). Chronic ethanol feeding decreased the cytoplasm profile area, as well as the total number of cytoplasm granules per cell profile (Fig. 4). There were no significant differences between the results with respect to the ratio of azurophilic to specific granules among the 4 groups. There were changes in the topographic distribution of granules such as cytoplasmic areas with numerous granules and areas with a smaller number of granules or without them in the ethanol fed rats (Fig. 4). Some PMNs obtained from alcohol fed rats had autophagic vacuoles (Figs 5a and b).
As shown in Table IV, in the alcohol fed rats (group III) most of the neutrophil functions tested were unaffected. However, there was a depression of chemotactic activity (both spontaneous migration and chemotactic response) and an increase in absolute index of ingestion. On the contrary, protein deficient animals (group II) showed a significant (P
Discussion
The present study demonstrates that the rats given a chronic dose of alcohol consumed adequate amounts of other nutrients and had a normal growth rate and body weight at the end of the experiment. Therefore, this ethanol diet was nutritionally adequate and the effects due to alcohol and malnutrition were not confounded. Treatment of rats with ethanol resulted in a significant increase in the number of PMNs. It has been reported5 that ethanol inhalation altered the relative proportion of lymphocytes and PMNs in the peripheral blood. A factor that may be important with respect to this phenomenon is adaptation to the stress of ethanol ingestion in long-term studies such as these. In addition, an earlier study from our laboratory showed a significant increase in the granulocyte-macrophage committed precursor (CFU-GM) compartment in the animals treated with a single heavy dose of ethanol6. The effect of incubation with alcohol in vitro on PMN lysozomal enzyme release in response to degranulation stimuli has not been adequately evaluated and enzyme concentrations have not been measured in PMNs exposed to alcohol in vivo.
In the present study, cationic bactericidal proteins cytochemical score was significantly decreased after chronic ethanol feeding. In addition, in these animals Sudan black B positivity score was increased which may represent disturbance of the phospholipid composition of granules and vesicle membranes and granular matrix content. To the best of our knowledge there have been no studies of neutrophil cationic bactericidal proteins and Sudan black B positivity scores with chronic ethanol intake.
The present study also showed that PMN-phagocytosis and NBT reduction were unaffected by chronic alcohol exposure but granulocyte chemotaxis was impaired. A number of studies3,4,9 have shown that PMN-- ingestion and intracellular killing of bacteria are not influenced either in vitro or in vivo by exposure to the high concentration of ethanol. In vivo effect after chronic intoxication include dose-dependent impaired granulocyte chemotaxis10,11, depressed delivery to sites of inflammation and decreased adherence with high blood alcohol levels. Defect of stimulated movement of PMNs was detected in 60 per cent of patients with alcoholic liver disease11 . Aggregation responses of patients' PMNs suggest that the cellular defect may be related to specific abnormalities in the response to the CSa chemotactic factor11. It has been reported that in vivo neutrophil delivery in humans with alcohol cirrhosis was normal despite depressed in vivo chemotaxis'. In addition, among several PMN-properties tested in healthy volunteers, only phagocytosis was found to be significantly depressed after single large dose of alcohols. These data point to the limited effect of occasional alcohol consumption on the neutrophil behaviour. These findings also suggest that factors other than alcohol could be involved in the marked PMN dysfunctions observed in chronic alcoholics.
At present, result relating to the effect of protein malnutrition on PMN and mononuclear phagocytes are inconclusive. Our previous studies indicated that the activity of many PMN enzymes and antibacterial and bactericidal systems were altered in PMNs during protein malnutrition12,16,17 and this agrees with other reports that used PMNs as an indicator in experimental animals and several malnourished children's.
The attachment and ingestion of microorganisms by PMNs in the presence of serum is mediated by immunoglobulin Fc receptor and CR1 and CR3 complement receptors. It is possible that these receptors decrease in number or activity with malnutrition29. Our results regarding decreased NBT reduction in protein malnourished rats are in agreement with the observations by others in PMNs, monocytes and macrophages29. To the best of our knowledge, quantitative analysis of the fine structure of PMNs has not been studied in protein malnutrition. The electron microscopic studies on resting and phagocytosing phagocytes showed no qualitative differences in the granules, mitochondria, phagocytic vacuoles or extent of degranulation in children with kwashiorkor14. Our ultrastructural findings in PMN are not in agreement with these findings.
Our results showed that damage to PMNs induced either by alcohol or by protein malnutrition alone was of a similar degree. However, the damage was more severe for some parameters in animals that suffered simultaneously with malnutrition and alcoholism.
Acknowledgment
This research was supported by a grant from the Serbian Ministry of Science and Technology.
References
Borregaard N, Lollike K, Kjeldsen L, Sengelov H, Bastholm L, Nielsen MH, et al. Human neutrophil granules and secretory vesicles. Eur J Haematol 1993; 51: 187-98. Cassatella MA. The production of cytokines by polymorphonuclear neutrophils. Immunol Today 1995;16: 21-6. Ballard HS. Haematological complications of alcoholism.
Alcahol Clin Exp Res 1989; 13: 706-20. MacGregor RR. Alcohol and immune defense. JAMA 1986; 256: 1474-9.
Marietta CA, Jerrells TR, Meagher RC, Karanian JW, Weight FF, Eckardt MJ. Effects of long-term ethanol inhalation on the immune and hematopoietic systems of the rat. Alcohol Clin Exp Res 1988; 12: 211-4.
Stojanovic N, Budec M, Jovcc G, Bugarski D, Todorovic V. Effect of a single dose of ethanol on granulopoiesis in female rats: relationship to phase of estrous cycle. J Stud Alcohol 1996; 57: 344-8.
7. Swift RM, DePetrillo P. Human leukocyte beta-adrenergic stimulated cyclic AMP in ethanol intoxication and withdrawal. Alcohol Clin Exp Res 1990; 14: 58-62.
8. Corberand JX, Laharrague PF, Fillola G. Human neutrophils are not severely injured in conditions mimicking social drinking. Alcohol Clin Exp Res 1989; 13: 542-6.
MacGregor RR, Safford M, Shalit M. Effect of ethanol on functions required for the delivery of neutrophils to sites of inflammation. J Infect Dis 1988; 157: 682-9.
10. MacGregor RR. In vivo neutrophil delivery in men with alcoholic cirrhosis is normal despite depressed in vitro chemotaxis. Alcohol Clin Exp Res 1990; 14:195-9.
11. Rajkovic IA, Yousif-Kadaru AG, Wyke RJ, Williams R. Polymorphonuclear leukocyte locomotion and aggregation in patients with alcoholic liver disease. Clin Exp Immunol 1984; 58: 654-62.
12. Todorovic V, Koko V, Lackovic V, Milin J, Varagic J. Effect of chronic alcohol feeding on the ultrastructure of rat peripheral blood neutrophils. A morphometric study. J Stud Alcohol 1994; 55: 239-48.
13. Chhangani L, Sharma ML, Sharma UB, Joshi N. In vitro study of phagocytic and bactericidal activity of neutrophils in cases of protein energy malnutrition. Indian J Pathol Microbiol 1985; 28: 199-203.
14. Douglas SD, Schopfer K. Phagocyte function in protein-calorie
malnutrition. Clin Exp Immunol 1974;17:121-8. 15. Felsenfeld O, Gyr K. Polymorphonuclear neutrophilic leukocytes in protein deficiency. Am J Clin Nutr 1977; 30: 1393-7.
16. Todorovic V, Pavlovic M, Ristic M. The effect of hypoprotein nutrition upon granular cationic proteins and myeloperoxidase and lactic dehydrogenase enzyme activities in rat peripheral blood granulocytes. Study I. Acta Med lugosl 1986; 40: 57-70. 17. Todorovic V, Pavlovic M, Ristic M. Hypoprotein nutrition, phospholipid content and the phagocytic ability of rat peripheral blood granulocytes. Study II. Acta Med lugosl 1988; 42: 363-72.
18. Chandra RK, Kumari S. Nutrition and immunity: an overview. J Nutr 1994;124: 1433S-5S.
19. Palencia G, Teixeira F, Ortiz A, Perez R, Rios C, Sotelo J. Detrimental effects of malnutrition on the damage induced by alcoholism; a study of animal models that simulate chronic
alcoholism and malnutrition of large human groups. J Stud Alcohol 1994; 55:113-20.
20. Palencia G, Teixeira F, Ortiz A, Perez R, Sotelo J. Reversibility of the alterations induced by chronic alcoholism and malnutrition in rats after alcohol withdrawal and proper nutrition. J Stud Alcohol 1995; 56:140-6.
21. Salaspuro M. Nutrient intake and nutritional status in alcoholics. Alcohol Alcohol 1993; 28: 85-8.
22. Watzl B, Watson RR. Role of nutrients in alcohol-induced
immunomodulation. Alcohol Alcohol 1993; 28: 89-95. 23. Hayhoe FRJ, Quaglino D. Haematological cytochemistry, New York: Churchill Livingstone; 1980 p. 211-2. 24. Anderson DR. A method of preparing peripheral leucocytes for electron microscopy. J Ultrastruct Res 1965; 13: 263-8.
25. Graham RCJr, Karnovsky MJ. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 1966;14: 291-302. 26. Weibel ER, Kistler GS, Scherle WF. Practical stereological methods for morphometric cytology. J Cell Biol 1966; 30: 23-38.
27. Nelson RD, Quie PG, Simmons RL. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol 1975; IIS:1650-6. 28. Aleksejeva OG, Volkova AP. Phagocytic activity of neutrophils
in toxicological experiments. Gig San 1966; 8: 70-4. 29. De la Fuente M, Munoz ML. Impairment of phagocytic process in macrophages from young and old mice by protein malnutrition. Ann Nutr Metab 1992; 36: 41-7.
Reprint requests: Dr V. Todorovic, Institute for Medical Research, PO Box 721, Dr Subotica 4, I1001 Belgrade, Yugoslavia
Vera Todorovic Vesna Koko, Marijana Petakov, Gordana Jovcic, Neveka Stojanovic Diana Bugarski & Predrag Peric
Institute for Medical Research, Department of Experimental Hematology, Yugoslavia
Accepte February 1,1999
Copyright Indian Council of Medical Research Mar 1999
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