The effect of infection on leukocyte and serum lysosomal enzyme activities in protein-calorie malnutrition

NUTRITION RESEARCH, Vol. 9, pp. 15-26, 198g 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

THE EFFECT OF INFECTION ON LEUKOCYTE AND SERUM LYSOSOMAL ENZYME ACTIVITIES IN PROTEIN-CALORIE MALNUTRITION IN.I. Olomu, M.B.B.S.. F.M.C. Paed.; Ij.A. 0mene, M.D. and ~R.H. Glew, Ph.D. IDepartment of Child Health, College of Medical Sciences, University of Benin, Benin City, Nigeria, and 2Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

ABSTRACT

Representative leukocyte and serum lysosomal enzymes, namely B-glucuronldase, acid phosphatase and hexosaminidase, were studied in 53 protein-calorle malnourished children and 26 controls. The leukocyte lysosomal enzyme activities were elevated in non-infected malnourished children while significant decreases in these enzymes were observed in the leukocytes of the infected malnourished group whose serum lysosomal hydrolase levels were also significantly elevated. These observed alterations in serum and leukocyte hydrolase activities may partially explain the impaired leukocyte microbicidal activity and the increased propensity for the development of endotoxic shock in children with protein-caloriemalnutrition.

Key words:

Infection, Leukocyte, Serum, Lysosomal, Hydrolases, PCM

INTRODUCTION

Children with protein calorie malnutrition (PCM) are susceptible to increased bacterial infections resulting in considerable mortality and morbidity (1-3). Studies of the host defense mechanism in malnutrition have focused mainly on the microbicidal capacity of the polymorphonuclear leukocytes (PMN). One of the crucial steps in phagocytosis is associated with the oxygen burst, an increase in metabolic activity and the production of antimicrobial agents, particularly hydrogen peroxide and superoxlde anion (02-). Furthermore, lysosomal hydrolases released by PMN following degranulation assist in the digestion of engulfed microorganisms. Thus, impairement in R202 or O2-formation by phagocytising leukocytes may interfere with the antimicroblal system of the white cells. In this respect, Seith and Chandra demonstrated subnormal bactericidal ability of polymorphoneuclear cells from fifteen undernourished children to Staphylococcus aureus (4), while

IAuthor's Correspondence Address: J. A. Omene, M.D., Department of Pediatrics, St. Lukes-Roosevelt Hospital Center, Amsterdam Avenue & ll4th Street, New York, New York 10025 15

16

N.I. OLOMUet a l .

Shopfer and Douglas reported reduced capacity of PMN from kwashiorkor children to inactivate Escherlchea coli, Staphylococcus aureus and Candlda albicans (5,6). The leukocyte hydrolases have antlmicrobial activity (7), which are active against a wide range of gram negative and gram positive bacteria (8), and recurrent bacterial infections are prominent features of clinical syndromes associated with some lysosomal enzyme deficiencies (9). Studies of leukocyte lysosomal enzyme activities in PCM are few in number and have yielded conflicting results. Selvaraj and Bhat (i0) found comparable leukocyte activites of acid phosphatase in malnourished and control children while Avila et al. (11) observed a decrease in acid cathepsin activity in marasmus and increased alkaline phosphatase activity in kwashiorkor. Apart from the interest which the lysosomal hydrolases have generated with regards to host defense mechanisms, some investigators (12) have hypothesized, on the basis of animal models, that the lysosomal enzymes may play a key role in the development of irreversible cellular changes in bacterial endotoxic shock. Because bacterial infections complicated by endotoxic shock are frequently encountered in malnourished children, we were stimulated to determine the activities of representative serum and leukocyte lysosomal hydrolases in PCM. Specifically, it was of interest to investigate the alterations of these enzymes in infected malnourished children in an effort to elucidate the pathophyslological basis for the increased morbidity and mortality observed in malnourished children with bacterial infection.

MATERIALS AND METHODS

The study population consisted of 24 infected malnourished children (ii marasmus, 13 kwashiorkor); 29 non-infected malnourished children and a third group consisting of 26 well-nourished children, 15 of whom were infected. Blood cultures, suprapubic aspiration of urine and stool cultures were obtained from all the malnourished children (N=53) and the 15 infected well-nourished subjects in addition to radiologlcal evaluation of the chest. Bacteriological evaluation of the cerebrospinal fluid was also carried out when this was indicated. The 29 non-infected malnourished children who served as controls for the malnourished infected children had no clinical or laboratory evidence of overt microbial infections on the basis of the screening procedures enumerated above. Children with tuberculosis were excluded from the study. The nutritional status was classified according to the Wellcome classification (13). The children were all aged 6 months to 3 years. Six ml of venous blood was drawn from each child after informed consent had been obtained from the parents. A 2.5 ml aliquot was placed in a clean test tube and allowed to clot for about 45 minutes before it was centrifuged; the serum was removed and stored frozen in a clean test tube at -43~ until assayed for various enzyme activities. Another aliquot ( 3.5 ml) of the venous blood was placed in an EDTA bottle for determination of hematocrits and

LEUKOCYTE & SERUMLYSOMAL ENZYMES

17

total white cell counts. The remaining sample was used for isolation of leukocytes by the dextran sedimentation method and hypotonic lysing of red cell contaminants (14). The final leukocyte pellet was suspended in 1.5 ml physiologic saline and stored frozen at -43~ Before enzyme assays, the suspension was thawed and sonicated at 10 m~ for i0 seconds on ice.

Enzyme assays:

Twenty ~i aliquots of the homogenates described above were used to determine leukocyte B-glucuronidase (15), acid phosphatase (16) and hexosaminidase (17) activities using appropriate substrates of 4-methylumbelliferyl derivatives. Assays were carried out in duplicate in I0 x 75 mm borosilicate glass tubes in a final volume of 0.i ml, containing 3.0 mM substrate, 0.2 M sodium acetate buffer (pH 4.54) and the leukocyte homogenate. At the end of a 15 minute incubation at 37~ reactions were stopped by addition of 2.9 ml glycine-ammonium hydroxide buffer, pH 10.5. Fluorescence was determined using a Turner fluorometer (model III). One unit of enzyme activity is defined as the amount of enzyme that catalyses the hydrolysis of one nanomole of substrate per hour. Serum enzyme assays were performed using 20 ~i of serum as the source of enzyme.

Protein assay:

Total serum protein concentration and the protein content of the leukocyte homogenates were determined by the method of Lowry et al. (18) using bovine serum albumin as standard.

Statistical analysis:

The mean and standard error of the mean were calculated for all data and tests of statistical significance done by the Fischers student 't' test.

RESULTS

i.

Characteristics of the study population

(Tables I, 2 and 3)

The three groups of children had comparable mean ages, and the wellnourished controls had a mean weight appropriate for their mean age. The mean weights of the marasmic and kwashiorkor children were below 60% and between

18

N.I. OLOMU et al.

60-80%, respectively, of the 50th centile of their expected weight for age. The total serum protein concentration of the well-nourished children was significantly higher than those for the two groups of malnourished children (Table I).

TABLE I Mean Age, Weight and Total Serum Proteins of the Malnourished and Control Children Studied Well-nourlshed n = 26 (mean • SE)

Mean age (months)

20.5•

Mean weight (kg) Total serum proteins (gm/dl)

11.3• 7.2•

Kwashiorkor n = 31 (mean • SE)

Marasmus n = 22 (mean • SE)

18.0• n/s 7.7• P<0.001 5.7• P<0.001

16.5• n/s 6.1• P<0.001 6.3• P<0.005

P values for well-nourished versus the malnourished groups of children; n/s, not significant.

The pattern of infection was similar in the well-nourished and malnourished children (Table 2). Bronchopneumonia, which was the commonest infection in all groups, responded promptly to conventional antibiotic therapy. All of the various groups of children had comparable total leukocyte counts but the kwashiorkor non-lnfected children had significantly lower hematocrits than controls (Table 3).

TABLE 2 Pattern of Infection in the Groups of Children Studied Acute infection Bronchopneumonia Septicaemia Tonsillitis Otitis media Meningitis Abscesses Infective diarrhea

Controls

*Kwashiorkor

Marasmus

7 2 2 I 2 1 -

I0 3 1 -

6 1 I I 2

*One child had septicemia and bronchopneumonia

LEUKOCYTE & SERUMLYSOMAL ENZYMES TABLE 3 Hematocrit

and total leukocyte counts in wellnourished and PEM children Haematocrit ( % ) (mean t SE)

Total leukocyte count (xl09/e) (mean t SE)

Well-nourished Infected Non-infected*

15 Ii

32,6ti.9 35.3•

i0. i~i.i 9.5t0.8

Kwashiorkor Infected Non-infected*

13 18

29.1tI.6 28.4•

i0.7tI.4 10.9•

Marasmus Infected Non-infected

11 ii

31.3t2.4 32.9~1.5

ii.9ti.8 12.5•

*P value compares non-infected kwashiorkor children (P<0.02); N, number of subjects.

2.

Leukocyte lysosomal enzyme activities

and non-infected well-nourished

(Table 4).

(a) In the absence of infection: In the absence of overt infection, increased leukocyte lysosomal enzyme activities were observed in malnourished children compared with controls: the observed increases in the kwashiorkor children were 1.8-fold (P<0.05) and 2.0-fold (P<0.025) for acid phosphatase and hexosaminidase activities respectively, while in the marasmic children, the increases were 2.4-fold (P<0.01); 2.0-fold (P<0.02) and 2.6-fold (P<0.02) for B-glucuronidase, acid phosphatase and hexosaminidase respectively when these values were compared with controls. (b) In infection: The leukocyte enzyme activities were comparable in infected and non-infected well-nourished children. On the other hand, the infected malnourished children exhibited significantly lower activities than their non-infected counterparts. In the marasmic children, there was a decline of 65% (P<0.O05), 46% (P
19

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DISCUSSION

The present study shows that marked differences exist between the activities of leukocyte lysosomal hydrolases in well-nourlshed and malnourished children. This finding is consistent with those reported by Avila et al. (11) who found elevated acid phosphatase activity in leukocytes of kwashiorkor and marasmic children. Similarly, recent studies from our laboratory on small-for-date infants who are prototypes of prenatal malnutrition revealed significant increases in leukocyte B-gluruconidase and acid phosphatase activities (19). A possible explanation for the increased leukocyte enzyme activities in malnutrition may be related to the membrane stabilizing effect of cortisol (20), the plasma level of which is elevated in both small-for-date babies and in severe PCM (21). Another significant observation in the present study was that infection in malnourished children is associated with decreased leukocyte lysosomal enzyme activities. This finding may partly provide an explanation for the observed impairment of leukocyte microbicidal capability in PCM. While the explanation for the reduced leukocyte enzyme activity in infection cannot be deduced from this study, it may be related to the membrane destabilizing effect of bacterial toxins as was demonstrated by Weissmann and Thomas (21); and Janoff and coworkers (12). Both the lysosomal and the leukocyte plasma membranes in severely malnourished children may be unduly sensitive to bacterial toxins. Excessive release of lysosomal enzymes from the leukocyte would lead to their non-availability within the PMN for intracellUlar bacterial killing and digestion, with resultant increased morbidity and mortality from bacterial infections. This speculation is supported by the

22

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LEUKOCYTE & SERUM LYSOMALENZYMES

23

work of Alexander (22) who showed that the levels of leukocyte acid hydrolases are decreased in patients with severe burns. The lowest levels were observed between the sixth and tenth post-burn day; this coincided with the peak of mortality resulting from septic complications. An important question that needs to be addressed however, is why malnourished children with elevated leukocyte hydrolase activity, specifically acid phosphatase, should be excessively prone to infection. Several explanations seem plausible: first, it is possible that certain invading microorganisms possess the ability to inhibit the phagosome-lysosomal fusion in malnourished children. Indeed, Horwitz (23), showed that live Le$ionella pneumophila inhibit the fusion of phagosomes with lysosomes of monocytes, thereby providing an important mechanism for the survival of this organism within phagocytes. Furthermore, it has been demonstrated in more recent studies (24,25) that parasites such as Leishmania donovani and Le~ionella micdadel produce acid phosphatases capable of blocking the oxygen burst, thereby inhibiting the production of superoxide anions and hydrogen peroxide by stimulated neutrophils and promoting the survival of these parasites within the host cells. It could be argued however that no organism among our infected population was shown to exhibit the capability of blocking superoxide anion production. Nevertheless, studies by Das and coworkers (26) showed that endogenous mammalian prostatic acid phosphatase was very effective in suppressing the generation of superoxide anions. It is therefore conceivable that in pathological situations such as those found in PCM, increased activity of leukocyte acid phosphatase, as was found in the current study, may actually interfere with the generation of toxic oxidative radicals by phagocytes. Such a a situation will then present a favorable milieu for rapid bacterial growth in an already compromised host. The other important clinical observation in this study was the flnding that infected well-nourished children had comparable or lower serum lysosomal enzyme activities when compared with their non-lnfected counterparts, while the infected malnourished children had significantly higher serum enzyme activities than their non-infected counterparts. Furthermore, the infected malnourished children also had significantly higher serum enzyme activities than the infected well-nourished children. This observation may be important clinically because increased serum lysosomal enzyme activities have been linked with self-destructlve processes such as anaphylaxis (27) and shock (28), which may be associated with bacterial infections. Of interest in this regard, is the direct evidence derived from studies in animal model which suggest that the lysosomal hydrolases, at least in part, mediate the phenomenon of bacterial endotoxemia. Weissmann and Thomas (21) showed that cathespin and B-glucuronidase are readily released from large granule fractions of rabbit liver in vitro when the young rabbits were injected with bacterial endotoxin. Janoff and Coworkers (12), extending this study, demonstrated that endotoxic shock caused increased release of lysosomal enzymes, specifically B-glucuronidase and acid phosphatase, into the circulation -- a process that was intimately associated with tissue injury and lethal progression of the pathological events observed in bacterial endotoxemia. Interestingly, Saito and Suter (28) also demonstrated a positive correlation between the degree of sensitivity to the lethal effects

24

N.I. OLOMUet al.

of endotoxin and the release of lysosomal enzymes from the tissues into the serum in experimental animals. It is therefore reasonable to suggest that the finding of elevated serum lysosomal enzyme activities in infected malnourished children may partially explain the clinical severity of bacterial infections complicating severe protein-caloric malnutrition. Furthermore, the decreased leukocyte lysosomal enzyme activity observed in this high risk group of children may be an important contributory factor for the high mortality associated with PCM. In summary, we have demonstrated elevated leukocyte lysosomal enzyme activities in non-infected malnourished children. Infected malnourished children have lower leukocyte enzyme activities than their non-infected counterparts. In contrast, however, significantly higher serum lysosomal enzyme activities were observed in infected malnourished children. These alterations in leukocyte and serum enzyme activities may play a vital role in the increased mortality and morbidity of malnourished children whose clinical course is often complicated by bacterial infections.

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21

Suskind RM.

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21.

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N.I. OLOMUet al.

25.

Remaley AT, Glew RH, Kuhn DB, Basford RE, Waggoner AS, Ernst LA, Pope M. Leishmanla donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production. Exp Parasltol, 1985. 60:331-41.

26.

Das S, Saha AK, Remaley AT, Glew RH, Dowling JN, Kajiyoshi M, Gottlieb M. Hydrolysis of phosphoproteins and inositol phosphates by cell surface phosphatase of Leishmania donovani. Mol Biochem Parasitol, 1986; 20:143-53.

27.

Movat HZ, Uniuhara T, Taichman NS. The role of polymorphonuclear leukocyte lysosomes in tissue injury, inflammation and hypersensitivity. VI. The participation of the PMN leukocyte and blood platelets in systemic aggregate anaphylaxis. Immunology, 1968; 14:637-48.

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Accepted for publication August 29, 1988.