Ultrastructural and morphometric analysis of thymic epithelial secretory vacuoles in severely protein-energy malnourished weanling mice

NUTRITION RESEARCH, Vol. 6, pp. 663-671, 1986 0271-5317/86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

ULTRASTRUCTURAL AND MORPHOMETRIC ANALYSIS OF THYMIC EPITHELIAL SECRETORY VACUOLES IN SEVERELY PROTEIN-ENERGY MALNOURISHED WEANLING MICE

Alpana Mittal, M.Sc. and Bill Woodward, Ph.D. Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ontario NIG 2WI Canada

ABSTRACT

The ultrastructure of hormone-secreting vacuoles of thymic stellate epithelial cells was examined in the two most common rodent models of protein-energy malnutrition viz. animals fed a nutritionally adequate diet in restricted Quantities (R mice) and animals fed ad libitum a low-protein formulation (LP mice). Male and female CBA/J mice including an ad libitum-fed control group (C mice) were fed from 23 to 37 days of age. Both R and LP mice developed severe malnutrition as indicated by weight loss, low serum protein levels, low thymic and splenic indices, lymphocyte depletion from the thymus and low (LP mice) or high (R mice) maximal hepatic alanine aminotransferase activity. Lipid-laden stellate epithelial cells devoid of hormone-secreting vacuoles were found in the thymic cortex and medulla of the malnourished animals. In addition other stellate epithelial cells in the cortex exhibited enlarged vacuoles which were fewer in number than those of C mice and many of which were concentrated in the perinuclear zone. In R mice vacuoles were not found in the peripheral cytoplasm and frequently exhibited increased quantities of electron-dense content, possibly hormonal material. The results suggest impaired hormone secretory activity by the thymic epithelium of both R and LP mice. KEY WORDS:

protein-energy malnutrition,

mice. thymus

INTRODUCTION

Immune responses mediated by thymus (T)-lymphocytes are impaired in severe protein-energy malnutrition (PEM) (I). Associated with this general finding is a selective depletion of mature T lymphocytes from the blood and secondary lymphoid organs in malnutrition (I). Thymie epithelial cells appear to synthesize factors required for T-lymphocyte maturation (2-4), and the serum level of one such factor, the peptide FTS, is low in malnourished children (5) and rats (6). Brief incubation of peripheral blood cells with thymic hormones induced a normal proportion of mature T lymphocytes to appear in samples from malnourished children (7). Moreover, injection of thymic hormones improved cell-mediated disease resistance in protein-deficient mice

663

664

A. MITTAL and W. WOODWARD

(8). The foregoing discoveries suggest that a primary effect of PEM on the immune system may lie at the level of the thymus epithelium, and direct evidence of abnormalities in this tissue has been obtained by the authors in studies of severely food intake-restricted weanling mice (9). Our hypothesis is that PEM impairs the ability of the thymus to replenish the depleted immune system in a manner analogous to the effect of thymectomy on sub-lethally irradiated adult rodents. In the present work, mice fed a low-protein diet were used in addition to animals fed a nutritionally adequate diet in restricted quantities. Thus the present investigation included the two most common rodent models of severe PEM. The main objective of this study was to examine, in both models of PEM, the ultrastructure of the thymic epithelial vacuolar system which is considered to function in hormone secretion (3)- Accordingly the present investigation was directed to the stellate epithelial cells which are found in both the cortex and the medulla of the thymus (10,11).

MATERIALS AND METHODS

Animals, diets and dietary treatments. Male and female weanling (21-day-old) CBA/J mice were individually housed in plastic cages. Room temperature was about 24~ and a photoperiod of 14 hours light and 10 hours darkness was maintained. All animals were acclimated for 2 days on the nutritionally adequate control purified diet (Table I). The mice had free access at all times to clean tap water, and the experimental feeding period was 14 days i.e. from 23 to 37 days of aEe. Control (C) animals were fed add libitum the diet shown in Table I, and food intake restricted (R) mice were fed the same diet in restricted daily portions as previously described in detail (9). Briefly, R mice were fed, for the first 9 days, 50~ of the a~erage pre-determined ad libitum intake of C mice (g feed/g live weight day- ), followed by 60% of--a_9_d libitum intake for the last 5 days. A second group of malnourished mice, designated low-protein (LP) animals, was fed ad libitum a diet which contained 0.3% crude protein by Kjeldahl analysis (12). This diet was made from the formulation shown in Table I by weight-for-weight replacement of egE white with cornstarch. Measurements and analytical procedures. Initial and final live weiEhtS were recorded together with the daily food intake of each mouse. At the end of the 14-day feedin E period serum protein levels, thymic and splenic indices (orEan weight per unit live weiEht) and liver maximal alanine aminotransferase activities were determined as described previously (9) on separate subEroups within each of the three treatments (C, R and LP). Thymuses from 5 C, 10 R and 14 LP mice were prepared as indicated elsewhere (9) for light microscopy. At least five wax sections were examined per animal. In addition, thymuses of 9 C, 16 R and 6 LP mice were prepared for electron microscopy as previously outlined (9), and at least four sections per animal were examined. Location within the orEan (i.e. cortex or medulla) was determined primarily on the basis of lymphocyte numbers in I ~m sections stained with toluidine blue. The corticomedullary junction, recognized by its larEe blood vessels (11), was included with the medulla in the present study. Thymic stellate epithelial cells were identified ultrastructurally by the presence of desmosomes or tonofllaments which are characteristic features of these cells (10,11,13). Morphometric analysis of electron microEraphs from 7 C mice, 6 LP mice and 6 R animals provided estimates of the

THYMUS IN MALNUTRITION

665

Table 1 Composition of purified diet

Ingredient

I

Amount (g/kg)

Spray-dried egg white 2

245

Cornstarch 3

290

Glucose

4

290

Cellulose 5

50

Corn oil 6

75

Vitamin-mineral supplement 7

50

Iproximate analysis (% as-fed basis): d r y matter, 92.3; crude protein (% N x 6.25), 19.2; ether extract, 7.9; crude fiber, 3.8; ash, 4.5; gross energy, 217.2 kJ/g (4.1 kcal/g). Methods according to the AOAC (12). ~U.S. Biochemical Corp., Cleveland, Ohio. ~SL. Lawrence Starch Company, Ltd., Port Credit, Ontario. _Crelose, 2001, CPC International, Englewood Cliffs, N.J. Celufil, nonnutritive bulk, U.S. Biochemical Corp. Contained sufficient supplemented fat-soluble vitamins to provide per kilogram diet: 1000 IU cholecalciferol; 50 IU all-rac-a-tocopheryl acetate. The supplement supplied the following levels of nutrients per kilogram diet: 4000 IU retinyl acetate; 0.8 mg menadione sodium bisulfite; 4 m E biotin; I mg folic acid; 15 mg niacin; 12 mg D-calcium pantothenate; 10 mg riboflavin; 8 mg thiamin HCI; 9 mg pyridoxine HCI; 0.05 mg cyanocobalamin; 1.2 mg choline chloride; 2.5 g NaCI; 27.8 g dicalcium phosphate (20% Ca, 18% P); 0.9 g maEnesnium oxide (60% ME); 10.7 g potassium sulfate (45% K); 21.6 mg cupric sulfate (25% Cu); 174 mg ferrous sulfate (21% Fe); 154 mg manganous sulfate (25% Mn); 0.3 mg potassium iodide (75% I); 0.4 m E sodium selenite (30% Se); 14 mg zinc carbonate (52% Zn). The supplement was brought to appropriate weight with glucose.

~

number of secretory vacuole profiles pep ~m 2 of cortical stellate epithelial

cell section area. Quantification of this parameter, designated N. (14), required point counting analysis by means of a regular quadratic l~ttice with 1.00 cm spacing. Print magnification of 8316 X was determined from photographs of a standard grating r e p ~ c a and each lattice point, therefore, represented a section area of 1.44 ~m . Vacuolar size was estimated by direct measurement of profile diameters. Minimal sample size for each morphometric parameter (vacuolar NA and profile diameter) was determined as previously described (15) and, f~r each mouse, the sample obtained on both parameters was at least twice the minimal. Statistical analyses were performed according to Steel and Torrie (16). The 5% level of probability was considered sufficient to demonstrate statistical differences.

666

A. MITTAL and W. WOODWARD RESULTS AND DISCUSSION

During the 14-day experimental period, C mice Eained more than 70% of their initial weiEht while R and LP mice lost about 30% and 20%, respectively (Table I). ~ood intakes (X+SD) were 43.7+3.6, 14.6+2.8 and 30.0• E/mouse 14 days- for the C, R and LP groups ~espective~y. The severity of malnutrition imposed on the R and LP mice is further indicated by their reduced thymie and splenic indices and low serum protein levels (Table I), phenomena commonly observed in severe PEM (I). Of 47 mice initiated on the R protocol, 9 were lost throuEh mortality between the ninth and fourteenth days of the experiment. No mortalities occurred within the LP (or the C) Eroup. Both LP and R mice exhibited reduced numbers of thymic cortical lymphocytes per unit of tissue area examined histologically. This is a common feature of PEM in experimental animals and human beinEs (I). The effect was particularly severe in R mice which also exhibited lower thymie indices than LP mice (Table I). Although the serum protein values indicate protein deficiency in both malnourished groups, the alanine aminotransferase results (Table I) suggest, as is reasonable to expect, that protein was a more important enerEy source for R mice than for LP (or C) animals. Liver alanine aminotransferase is a key EluconeoEenic enzyme (17) which exhibits increased maximal activity (wet weiEht basis) when dietary energy is the primary limitinE factor (18) but decreased maximal activity when protein is the main limiting factor (18). In this reEard the Eross enerEy intake of LP mice

TABLE 2 Live WeiEhtS, Serum Protein Levels, Thymic and Splenic Indices, and Liver Maximal Alanine Aminotransferase Activities

Parameter

1

EMS 2

C

Group of mice LP

Initial liveweight (E/mouse)

11.0 a (20)

10.1 b (38)

Final liveweight (g/mouse)

18.9 a (20)

8 . 3 b (38)

7 . 6 c (30)

1.9

Serum protein level (E/100 ml)

5 . 4 a (9)

3 . 4 b (13)

3 . 3 b (14)

0.1

Thymic index (mE/E live wt.): a) Males b) Females

4

1.3 D (13)

(14)

0.1 0.1

Splenic index (mE/E live wt.)

3 . 2 a (11)

1.4 b (38)

1.1 c (26)

0.1

I0.8b (12)

64.0c (18)

263.2

Maximal liver ALAT 3 activity (pmo~e NADPH oxidized/E wet wt. min- )

(5)

42.2a (9)

R

10.5 a ' b

(30)

IMean value (number of mice). Within the same row, values not sharin E a superscript letter are different by Duncan's New Multiple Range Test. Error mean square. Alanine aminotransferase.

~

2.6

THYMUS IN MALNUTRITION

667

exceeded that of C mice during the 14-da~ feeding period (4.3• (n:21) vs. 3.6• (n=20) kd/g live weight day- , P<0.O01, two-tailed Student's ttest), while the caloric intake of R mice was approximately one-half that of the controls (9). Overall, therefore, these results support the expectation that LP and R mice represent metabolically dissimilar models of severe protein-energy malnutrition.

TABLE 3 Size and Number of Secretory Vacuoles in Cortical Stellate Epithelial Cells

Parameter C

Profile diameter

(pm)

Group of mice i LP

EMS2 R

0.76 a (7)

1.09 b (6)

1.86 c (6)

0.05

NA (~m-2) 3

0.31 a (7)

0.11 b (6)

0.04 b (6)

0.01

Nv ( p m - 3 ) 4

0 . 3 2 a (7)

0.08 b (6)

0.02 b (6)

0.02

IMean value with number of mice in parentheses. Values within the same row but not sharing a superscript letter are different by Duncan's New Multiple 2Range Test. RError mean square. ~Number of vacuole profiles per square micron of epithelial cell section area. -Number of vacuoles per cubic micron of epithelial cell volume. Derived from N. and average profile diameter according to the procedure of DeHoff and R~ines (1961) described elsewhere (14).

The abundant vacuoles of cortical and medullary stellate epithelial cells in C mice (Fig. la) exhibited the morphology and apparently random cytoplasmic distribution previously described (I0,11,13). Epithelial ceils with vacuolar morphology similar to that of C mice were common in the medulla of LP mice and less frequent in the cortex of these animals. By contrast, normal vacuolar morphology was never observed either in medullary or in cortical epithelial cells of R animals. R mice exhibited numerous cortical stellate epithelial cells in which the vacuolar apparatus was confined to the perinuclear region (Fig. Ib). These vacuoles were larger but fewer in number than the vacuoles of C mice (Table 3), and contained a greater quantity of granular, electron-dense material. LP mice also exhibited cortical stellate epithelial cells with reduced numbers of vacuoles compared to C mice, and these structures were intermediate in size between the vacuoles of the R mice and the C animals (Table 3). Although perinuclear accumulations of vacuoles were found in the cortical epithelium of LP mice, many vacuoles were also seen throughout the cytoplasm in this group of malnourished animals (Fig. Ic). Moreover the vacuolar content displayed normal fine structure in LP mice (Fig. Ic). Epithelial cells displaying the indicated abnormalities in vacuolar content and/or size and cytoplasmic distribution were particularly numerous in the subcapsular region of the malnourished animals, were seen in decreasing frequency towards the deeper regions of the cortex, and were not seen in the

668

A. MITTAL and W. WOODWARD

FIG. I Cortical stellate epithelial cells labelled to show nucleus (N), desmosome (D), tonofllaments (T) and vacuoles (V) including apparent vacuolar fusion events (F), x 6930. Circled structures shown within insets, x 23760. Tissue from (a) well-nourished, (b) R and (c) LP mice.

THYMUS IN MALNUTRITION

669

medulla. Finally LP mice, only, exhibited medullary epithelial profiles with numerous cytoplasmic vesicles about 0.1 ym in diameter. When apparent, these structures were seen together with a morphologically normal vacuolar apparatus. Their relationship to the vacuolar system is unknown. Thymic epithelial vacuolar glycoproteins appear to arise from the Golgi apparatus (10) so that, by implication, the vacuoles are Golgi-derived. The observed perinuclear accumulations of vacuoles therefore suggest impaired peripheral migration of these structures in cortical stellate epithelial cells of severely malnourished mice. Such an impairment in directed mobility could result from a cytoskeletal defect (19-21) and impair the secretion of hormones from the cortical epithelium. The large vacuoles in the cortical epithelium of the malnourished animals may result from fusion events secondary to reduced peripheral movement in a manner analogous to the macrovacuolation of fibroblasts cultured in high levels of cytochalasin D (20,21). Numerous vacuolar profiles in the malnourished mice displayed evidence of fusion in progress at the time of tissue fixation (Fig. Ib,c). The R mice may represent an extreme degree of pathology such that no peripheral vacuoles occur and "hormonal material accumulates to appear, as seems possible from the discussion of Nabarra et al. (22), as an increased quantity of electron-dense vacuolar content. No explanation can be given presently for the observation that these vacuolar abnormalities were confined to the thymic cortex. It is of interest, however, that thymic cortical and medullary stellate epithelial cells, although both ectodermal in origin (23), differ in pre-natal and post-natal development rate (24) and, at maturity, differ in their complements of surface antigens (25) as well as in the nature of their hormonal content (2,3). As described previously (9) cortical and medullary epithelium of R mice exhibited lipid-laden stellate cells devoid of secretory vacuoles. LP animals in the present study displayed similar abnormal stellate epithelial cells, presumably inactive in the release of peptide hormones. The lipidic nature of the cytoplasmic droplets in these cells was demonstrated in the present study by ultrastructural comparison of tissue fixed with and without osmium as previously described for thymuses of food-restricted mice (9). The lipid material is mainly esterified cholesterol in R mice (9), but its chemical identity in LP animals was not investigated in the present study. The present results suggest impaired function in the hormonesecreting apparatus of many thymic cortical stellate cells and absence of this subcellular apparatus from thymic cortical and medullary lipid-laden epithelial cells in the two most common rodent models of PEM. Low serum levels of FTS which occur in food-intake restricted rodents (6) and malnourished children (5) may also be expected, therefore, when low-protein (imbalanced) diets are fed. Moreover, although the thymic epithelium exhibits regional specificity in hormone content (2,3), it may be predicted that intrathymic and serum levels of all thymus hormones will be low in PEM. At present the influence of malnutrition on the quantity of thymic epithelium has not been quantified. Such information would aid in the interpretation of the present results and is currently being obtained in the authors' laboratory.

ACKNOWLEDGEMENTS

The authors are indebted to Mrs. Vera Peacock for conducting the enzyme assays. This work was supported by an NSERC individual operating grant given to B,W.

670

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