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In vitro studies of lymphocytes from children with kwashiorkor
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOCY
In Vitro Studies
5,21-30
(1976)
of Lymphocytes Kwashiorkor
from
Children
with
KURT SCHOPFER~ AND STEVEN D. DOUGLAS Nestle Foundation Research Center, Adiopodoume, Abidian, Ivory Coast, Departments of Medicine and Microbiology, University of Minnesota Medical Minneapolis, Minneapolis, Minnesota 5.5455
and School.
Received June 6, 1975 In vitro lymphocyte functions for children with kwashiorkor have been studied and compared to controls. Peripheral blood lymphocyte counts are similar to controls. Atypical mononuclear cells have been observed frequently in the peripheral blood smear. Electron microscopic observation revealed an increased number of plasmacytoid cells and occasionally lymphoblasts among isolated lymphocytes. In vitro response to phytohemagglutinin was decreased significantly, whereas pokeweed mitogen stimulation demonstrated higher response in 60% of the kwashiorkor patients studied and [3H]thymidine incorporation comparable to controls in 40%. The absolute number of rosette forming lymphocytes (T and B cell rosettes) was reduced in the kwashiorkor patients. Lymphocyte depletion of the thymus-dependent areas and reduction in the number of primary follicles and germinal centers were the main results of the histopathological examination of the thymolymphatic tissues. These findings suggest an acquired defect in cell-mediated immunity in kwashiorkor children.
INTRODUCTION
Increasing evidence for depression of the cell-mediated immune (CMI) response in children with protein-energy malnutrition (PEM) has accumulated since the relationship between malnutriton and infection has been recognized (l-3). Histopathologic findings, investigation of the in vitro lymphocyte function and in vivo testing, as well as clinical observations, suggest possible impairment of the thymus-dependent Q lymphocyte mediated immune reactions. Reports dealing with in vitro lymphocyte function have yielded conflicting results, which are probably attributable to the methodology employed or to a lack of clear definition of the syndrome involved. Therefore, the present study was undertaken to obtain further information about lymphocyte function of malnourished children. Accordingly, we have analyzed the rosette-forming ability, in vitro response to phytohemagglutinin (PHA-M) and pokeweed mitogen (PWM), and the electron microscopic features of lymphocytes from a group of children with kwashiorkor. PATIENTS AND METHODS
Twenty-seven children with a typical kwashiorkor syndrome admitted to the Pediatric Ward of the Centre Hospitalier de l’universite de Treichville, Abidjan, Ivory Coast, have been investigated and compared to a group of healthy control African children. Those with clinically overt infections were excluded from the study. Hematologic (hemoglobin, total leukocyte, and differential counts) and ’ Present address: Institute of Microbiology, Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
9008 St. Gallen, Switzerland.
22
SCHOPFER
AND
DOUGLAS
biochemical examinations (total protein, serum electrophoresis, cholinesterase activity) were performed by standard methods. Immunoglobulins (IgG, IgA, IgM) were measured by single radial immunodiffusion according to Mancini (4), using commercial immunodiffusion plates (Behringwerke, Marburg, Germany). Isolation of Lymphocytes The preparation of the lymphocytes was always performed within 2 hr after the blood samples were obtained. Lymphocytes were isolated from venous blood (containing nontoxic heparin, Upjohn, 20 units/ml) by the Ficoll-Hypaque density gradient method, as described by Boyum (5). The isolated cells were washed twice with phosphate buffered saline (PBS), pH 7.4, and suspended in the appropriate medium as indicated below. A differential count was performed and the lymphocyte number adjusted to the appropriate concentration. Rosette Formation Sheep red blood cell rosettes (SRBC-R) were prepared according to the method that we have previously described (6). In each preparation a minimum of 200 lymphocytes were counted in a hemocytometer and lymphocytes were designated rosette-positive if one or more SRBC’s adhered to the lymphocyte (“small” rosettes were defined as one or two adherent SRBC’s: “large” rosettes were lymphocytes with three or more attached SRBC’s.) Complement receptor lymphocytes (CRL) were assessed by the method of Michlmayr and Huber (7). The specimens were coded and identified after the percentage of rosette-forming cells was discerned. All preparations were examined by the same observer. Mitogen Stimulation Isolated lymphocytes were suspended in TC 199 containing 20% heat inactivated fetal calf serum (FCS) and adjusted with N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid (HEPES) to a pH of 7.4. PHA (Difco) or PWM (Gibco) was diluted in TC 199. Lymphocyte cultures were prepared that contained 3 x IO5 lymphocytes and 100 pg of PHA or PWM in a final volume of 0.3 ml per assay. Assays were performed in flat-bottom microculture plates (Falcon). Experiments were done in triplicate; samples without mitogen were always included. Cultures were incubated for 72 hr at 37°C. Eight hours before harvesting, 0.5 &i of [3H] thymidine (Amersham) were added. At the time of harvest, the incubation mixture was aspirated and filtered through a Millipore filter (1.2 pm pore size). The wells were washed repeatedly with PBS. The cell protein was then precipitated on the filter by successive washes with 10% followed by 5% trichloroacetic acid. The tilters were removed carefully, dried, and placed into a scintillation vial; 0.2 ml of 2 N KOH was added to dissolve the protein (8,9). After 30 min, scintillation fluid was added. The samples were counted in a liquid scintillation counter and the results were expressed as counts per minute. Electron Microscopy Isolated lymphocytes were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate containing sucrose. The samples were stored at 4°C. Cell preparations were postfixed in osmium tetroxide, dehydrated, and embedded in Epon. Specimens were stained en bloc with uranyl acetate. The samples were examined with a Siemens 102 electron microscope at an accelerating voltage of 80 V.
LYMPHOCYTES
IN
KWASHIORKOR
23
Histopathology Histopathologic examination of thymus, lymph node, spleen, and liver were performed for the single typical case in which it was possible to obtain autopsy material. RESULTS
Clinical and Laboratory Findings The children showed a typical kwashiorkor syndrome, as described earlier (10). The albumin levels (the main biochemical indicator of the degree of kwashiorkor) were always highly reduced (mean + SD): 1.85 ? 0.29 g/100 ml (n = 27) in comparison to controls: 3.88 + 0.45 g/100 ml (n = 23; p < 0.001). The total and differential leukocyte counts are summarized in Table 1. Higher neutrophil counts (p < 0.01) and a striking eosinopenia (p < 0.001) were encountered in the kwashiorkor children. There was no significant difference in the peripheral lymphocyte count. Atypical lymphocytes with a diminished nuclear/ cytoplasmic ration, increased basophilia, and cytoplasmic vacuolization were observed occasionally in the peripheral blood smear. Humoral Immunity Immunoglobulin levels are summarized in Table 2. No significant were observed between kwashiorkor children and controls.
differences
Rosette Formation SRBC-R. The total number of SRBC-R is lower in children with kwashiorkor @ < 0.05) than in the controls (Table 3). The percentage of rosette-forming lymphocytes, however, was similar in both groups. If lymphocytes to which three or more SRBC adhered were counted (“large rosettes”), then significantly fewer lymphocytes of kwashiorkor children formed rosettes QJ < 0.05). CRL. The absolute number of CRL was reduced for children with kwashiorkor (Table 3) @ < 0.05). There was no significant difference in the percentage of isolated lymphocytes binding to complement-coated SRBC. Mitogen Response PHA. The tritiated thymidine incorporation by lymphocytes from kwashiorkor children after 72 hr of incubation with PHA was diminished. The average values for the kwashiorkor patients were (mean + SD): 8917 + 6725 cpm (n = 15) compared to 24,149 f 8328 cpm (n = 28) for the controls. The difference is highly significant (p < 0.001) (Fig. 1). Cultures incubated without mitogens showed very low thymidine incorporation and there was no difference between kwashiorkor and control children. PWM. Determination of thymidine incorporation for lymphocytes incubated with PWM for 72 hr revealed two types of responses for cells from the kwashiorkor children. Lymphocytes from six children showed significantly increased thymidine incorporation (mean + SD: 12,516 + 2602) when compared to controls (mean f SD: 4613 + 2201; n = 9; p < 0.001). Cells from four of the 10 tested malnourished children showed values comparable to controls (Fig. 2). Electron Microscopy Pellets from Ficoll-Hypaque
preparations
revealed many typical small lympho-
33 ? 11 46 IL 13
2570 2 710 4380 k 2500 p < 0.01
-c 0.05
TABLE
p < 0.001
916 of:495 106 + 201
Absolute
IgG
1475 + 340” 1280 ? 130 ns.
(mg/lOO ml)
Total
62.3 t 7.1 57.2 ? 11.9 n.s.
.--Percentage
“Large”
w
2465 r 745 1147 2 531 < 0.05
65.3 t 10.9 32.1 t 10.4 c 0.05
“Small”
957 + 395 909 2 394 n.s.
Absolute
174 2 88 146 ? 39 n.s.
IgA
5*3 6~4
_--
17.0 r 6.6 75.1 t 5.3 i 0.05
Percentage
SRBC
tl.S.
131 ” 70 151 + 38
(mg/lOO ml)
340 _f 170 430 -t 300 ns.
Percentage
Monocytes Absolute
(mg/lOO ml)
LEVELS
Percentage
SRBC
SRBC-R (n =8)
Absolute
3
2
12 k 5 122
ROSETTE-FORMING ABILITY
TABLE
COUNTS
Percentage
Eosinophils
IMMUNOGLOBULIN
LYMPHOCVTE
1
LEUKOCYTE
TABLE BLOOD
’ All data mean t SD. * Student r- test; n.s., not significant.
3423 2 1043” 2055 or 747
Absolute
Ph
Controls (n = 14) Kwashiorkor (n = 18)
n All data mean -t SD. ’ Student t- test; n.s., not significant
Controls Kwashiorkor
P”
7576 I 2300 9066 + 4000 n.s.
Percentage
Neutrophils Absolute
” All data mean 2 SD. * Student r- test; n.s. = not significant.
Controls (28) Kwashiorkor (27) Significanceb
Total” Leukocytes
PERIPHERAL
4635 -t 1166 2727 ?z 1099 < 0.05
,4bsolute
14.6 ‘- 7.8 8.3 -c 4.2 n.s.
Percentage
_-_.-.--
50 t 9 47 2 11
Percentage
CRL (n =S)
3800 * 1600 4150 + 1900 n.s.
Absolute
Lymphocytes
w P
LYMPHOCYTES
IN KWASHIORKOR
25
-
Cuntrols
Kwashiorkor
FIG. 1. Comparison of the response to PHA of lymphocytes from children with kwashiorkor (n = 15) to controls (n = 28), cpm/3, lo5 lymphocytes. PHA Stimulation, 72 hr. (Individual cases; mean f SD)
cytes. There were frequent cytoplasmic lipid vacuoles, some cytoplasmic vacuolization, and occasional cells with increased cytoplasm and some increase in endoplasmic reticulum. About 10% of the cells seen had well-developed rough-surfaced endoplasmic reticulum in varying states of dilatation, features characteristic of plasmacytoid cells. However, plasmacytoid cells also were observed in some of the controls at a lower frequency. In addition, cells with large nucleoli and prominent euchromatin (lymphoblasts) were observed rarely in the kwashiorkor children (Fig. 3 and 4).
..
controls
Kwaohiorkar
FIG. 2. Comparison of the response to PWM of lymphocytes from children with kwashiorkor (n = 10) to controls (n = 9); cpm/3 x 105 lymphocytes. PWM Stimulation, 72 hr.
26
SCHOPFER
AND
DOUGLAS
FIG. 3. Electron micrograph of Ficoll-Hypaque mononuclear cell pellet from a child wi ith kwashiorkor. Note the lymphoblast with prominent nucleoli; several of the small lymphocytes also coIntain nucleoli. x 5100.
FIG. 4. Electron micrograph of plasmacytoid cell with dilated rough-surfaced re:ticulum from peripheral blood of kwashiorkor patient. x 14,000.
endoplasm lie
LYMPHOCYTES
IN
KWASHIORKOR
27
Histopathology Pertinent histopathologic findings include: marked thymic atrophy with difftculty in identification of typical Hassall’s corpuscles; depletion of the paracortical areas of lymph nodes and reduction in germinal centers, and primary follicles; depletion of the periarteriolar areas of the spleen; diffusely distributed lymphoblastoid cells with few discernible typical plasma cells and frequent occurrence of histiocytes in the sinusoidal areas of thymus, lymph nodes, spleen, and liver (Fig. 5 and 6).
FIG. 5. Spleen from autopsy of kwashiorkor tion. X 80.
patient. Periarteriolar
areas show lymphocyte deple-
FIG. 6. Thymic lobule from kwashiorkor patient. The lymphocyte depletion and paucity of readily identifiable Hassalls’ corpuscles are noted. x 80.
DISCUSSION
The present investigation of children with kwashiorkor showed normal peripheral blood lymphocyte counts, occasional atypical lymphocytes, decreased numbers of SRBC rosettes (T cells) and reduced numbers of CRL (B cells) when compared to controls. The response to PHA was diminished in the kwashiorkor children; tritiated thymidine incorporation with PWM incubation revealed two
28
SCHOPFER
AND
DOUGLAS
types of response (values comparable to controls in four children, significantly higher response in six children). About 10% of the isolated mononuclear cells showed plasmacytoid features by electron microscopy; blastoid cells were found rarely. The postmortem morphologic features of the lymphoreticular system included: severe thymic atrophy with absence of identifiable typical Hassall’s corpuscles, depletion of lymphocytes in the thymus-dependent areas of the lymph nodes and spleen, as well as decreased numbers of primary follicles and lack of germinal centers. Rather striking features were the presence of diffusely distributed lymphoblastoid cells and a prominent infiltration of the sinusoids of the lymphoreticular system with histiocytes. Studies by other investigators have revealed abnormalities ofin vitro lymphocyte function in malnourished children. Chandra (1 I) and Ferguson et ~1. (12) found decreased percentages of SRBC-R forming lymphocytes, but did not report absolute numbers. Reduced tritiated thymidine incorporation by lymphocytes following incubation with phytohemagglutinin has been demonstrated (13,14) and morphologic studies of blast transformation also showed diminished numbers of transformed cells with PHA (15-17). Our histopathologic findings are consistent with those reported by others (18-20). These observations may reflect a reduction in the circulating T cell pool size, changes in lymphocyte recirculation, and/or impaired differentiation or membrane abnormalities. Diminished PHA response and decreased rosette-forming ability are probably evidence for an abnormality of the circulating T cell population. The significance of “small” and “large” rosettes is unknown, yet it is possible that alterations in membrane properties are related to the observations of reduced numbers of “large” rosettes. Impairment of delayed hypersensitivity in malnourished children has been demonstrated in Go using PPD (21), DNCB ( 15, 22), Candida antigen (2), and keyhole limpet hemocyanin (KLH) (2). The increased incidence of infections and the unusual course of some of these infections (e.g., high incidence of measles giant cell pneumonia) (19,23) are further clinical evidence for abnormal CMI. The in vitro lymphocyte studies reported here and by others correlate with the abnormal CM1 suggested by clinical observations of these patients. Preliminary data suggest significantly lower absolute numbers of CRL (a B cell marker) in kwashiorkor children; however, the percentage ofCRL was not different compared to controls. The number of cases investigated is too small to draw definitive conclusions and other B cell markers must be studied. The presence of plasmacytoid cells in the peripheral blood is an unusual finding and has been reported in viral infections (24, 25) following thermal injury (26), in serum sickness (27), in Waldenstrom’s macroglobulinemia(28), in sarcoidosis (29), and after ingestion of berries or roots of Phytolucca americana (pokeweed) (30). The depletion of the B cell areas in lymph nodes and spleen, the observation of plasmacytoid cells in the peripheral blood, and the elevated response of lymphocytes from some patients to PWM could reflect the release of differentiated B cells into the circulation in kwashiorkor. The precise correlation between the morphology of lymphocyte-plasma cell development, mitogenic responsiveness, and surface markers is not yet known in clinical states.
LYMPHOCYTES
IN
KWASHIORKOR
29
Immunoglobulin levels are not different between kwashiorkor children and controls in our investigation. Reports dealing with the determination of immunoglobulin levels have shown conflicting results. Increased, normal, and reduced levels (3 I-35) have been found. The presence of infections, as well as the type and degree of malnutrition, must be considered in all evaluations. Our findings and those of others suggest that in a substantial proportion of kwashiorkor children, features of impaired CM1 are present, comparable to other clinical conditions characterized by defective CM1 function; yet the B cell dependent system in kwashiorkor children appears to be intact. Nevertheless, a number of unusual features remain to be unraveled. Immunodeficiency syndromes, either genetic or acquired, generally are associated either with peripheral blood lymphopenia (diminished numbers of B and/or T lymphocytes) and/or low immunoglobulin levels (36). Neither of these findings could be found consistently in kwashiorkor children. The lymphocyte depletion of the thymolymphatic tissues in kwashiorkor children is striking and it would be of importance of ascertain how normal, or only slightly reduced lymphocyte counts are maintained. Lymphocytopenia could be further expected by the hypercortisolemia (38, 39), which is always encountered in kwashiorkor children. Furthermore, the normal immunoglobulin levels remain to be explained. Reduction of the bone marrow-dependent B cell areas, few primary follicles, and germinal centers have been demonstrated in histopathologic examination and yet there is no reduction in immunoglobulins found. There is an increased preponderance of plasmacytoid cells in the peripheral blood, as well as an increased response to pokeweed mitogen, but this does not necessarily imply increased immunoglobulin synthesis, although hypergammaglobulinemia occasionally occurs in children with kwashiorkor. The situation is further complicated in kwashiorkor due to the hepatic steatosis and the severe liver disease with the possibility of impaired degradation of intestinal antigens. The role of the portal system in antigen presentation and antigen clearing has been investigated in patients with cirrhosis, where hypergammaglobulinemia and plasmacytoid cells are frequently observed (39,40). The role of this system in kwashiorkor should be further investigated. ACKNOWLEDGMENTS This work has been supported by the Nestle Foundation for the Study of the Problems ofNutrition in the World, the World Health Organization, and USPHSGrant AI-12478-01 from the National Institutes of Health. We thank Professors S. Badoual and N. Esso of the CHU Treichville for permission to study these patients. We are grateful to Sister Christine for her help. We thank Mr. J. F. Bois, Chief, Laboratory of Radioisotopes, ORSTOM, Abidjan, Ivory Coast for making scintillation counters available. We also appreciate the dedication and excellent technical assistance of Miss C. G. Mayer and Mrs. D. Schopfer.
REFERENCES 1. Scrimshaw, N. S., Taylor, C. E., andGordon, J. E., World Health Org. Mongr., Series 57, 1968. 2. Work, T. H., Ifekwunigwe, A., Jelliffe, D. B., Jelliffe, P., andNeumann, C. G.,Ann. Znt. Med. 79, 701, 1973. 3. Faulk, W. P., Demaeyer, E. M., and Davies, A. J. S., Amer. J. C/in. Nutrit. 27, 638, 1974. 4. Mancini, G., Carbonara, A. O., and Heremans, J. F., Immunochemistry 2, 235, 1965. 5. Boyum, A., Stand. J. Clin. Lab. Invest. 21, Suppl. 97, 1968. 6. Bentwich,Z.,Douglas, S. D., Siegal, F. P., and Kunkel, H. G., C’lin. Zmmunol. Zmmunopathol. 1, 511, 1973.
30
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AND
DOUGLAS
7. Michlmayr, G., and Huber, H., J. Immunol. 105, 670, 1970. 8. Douglas, S. D., Kamin, R. M., and Fudenberg, H. H., J. Immunol. 103, 1185, 1969. 9. Park, B. H., and Good, R. A., Proc. Nat. Acad. Sci. USA 69, 371, 1972. JO. Douglas, S. D., and Schopfer, K., Clin. Exp. Immunol. 17, 121, 1974. Il. Chandra, R. K., Brir. Med. J. 3, 608, 1974. 12. Ferguson, A. C., Law1or.G. J., Jr.,Newmann,C. G., Oh, W., and Stiehm, E. R.,J. Peds. 85,717, 1974. 13. Sellmeyer, E., Bhettay, E., Truswell, A. S., Meyers, 0. L., and Hansen, J. D. L., Arch. ZXS. Child.
47, 429, 1972.
14. Burgess, B. J., VOS, G. H., Coovadia, H. M.. Smythe, P. M., Parent, M. A.. and Loening, W. E. L., S. A. Med. .I. 48, 1870, 1974. 15. Smythe, P. M., Brereton-Stiles, G. G., Grace, H. J., Mafoyane, A., Schonland, M., Coovadia, H. M. Loening, W. E. K., Parent, M. A., and Vos, G. H., Lancer 2, 931, 1971. 16. Geefhuysen, J., Rosen, E. U., Katz, J., Ipp, T., and Metz, J., Brit. Med. J. 4, 527. 1971. 17. Grace, H. J., Armstrong, D., and Smythe, P. M., S. A. Med. J. 46, 402, 1972. 18. Mugerwa, J. W.,J. Pathol. 105, 105, 1971. 19. Schonland, M., Environ. Child. Hlth. 18, 217, 1972. 20. Watts, T., J. Trop. Peds. 15, 155, 1969. 21. Abbassy, A. S., Badr El-Din, M. K., Hassan, A. 1.. Aref, G. H., Hammad, S. A., El-Araby, 1. I.. and Badr El-Din, A. A., J. Trop. Med. Hyg. 77, 13, 1974. 22. Edelman, R., Suskind, R., Olson, R. E., and Sirisinha, S., Lancer 1, 506, 1973. 23. Bumet, F. M., Lancet 1, 610, 1968. 24. Farnes, P.. and Barker, B. E., Amer. J. Clin. Pathol. 49, 398, 1968. 25. Douglas, S. D.. Fudenberg, H. H., Glade, P. R., Chessin, L. N., and Moses, H. L., Blood 34,42, 1969. 26. Beathard, G. A., Granholm, N. A.. Sakai. H. A., and Ritzmann, !j. E.. C[in. Immuno[. Immunopathol.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
2,488,
1974.
Bamett, E. V., Stone, G., Swisher, S. N.. and Vaughan, J. H., Amer. J. Med. 35, 113, 1963. Zucker-Franklin, D., Sem. Hematol. 1, 165, 1964. Biberfeld, P., and Hedfors, E., Stand. J. Immunol. 3, 615, 1974. Farnes, P., Barker, B. E., Brownhill, L. E., and Fanger, H., Lance1 2, 1100, 1964. Samuel, A. M., Pate], B. D., and Mankodi, N., Indian J. Med. Res. 60, 1278, 1972. Rosen, E. U., and Geefhuysen, J., S. A. Med. J. 45, 980, 1971. Keet, M. P., and Thorn, H., Arch. Dis. Child. 44, 600, 1969. ElGholmy, A., Helmy, O., Hashish, S., Aly, R. H., and EI-Gamal, Y., J. Trop. Med. Hyg. 73,192, 1970. Aref,G. H., Badr El-Din, M. K.,Hassan, A. I., and Araby, 1. I., J. Trop. Med. Hyg. 73,186,1970. Cooper, M. D., Faulk W. P., Fudenberg, H. H., Good, R. A., Hitzig, W., Kunkel, H. G., Roitt, 1. M., Rosen, F. S., Seligmamt, M., and Soothill, J. F.,Clin. Zmmunol. Zmmunopathol. 2,416,1974. Beitins, I. Z., Kowarski, A., Migeon, C. J., and Graham, G. G., J. Peds. 86, 302, 1975. Godard, C., Helv. Pediat. Acta 29, 5, 1974. Bjomboe, M., Prytz, H., and orskov, F., Lancer 1, 58, 1972. Triger, D. R., Alp, M. H., and Wright, R., Lancer 1, 60, 1972.
IMMUNOLOGY
AND
IMMUNOPATHOLOCY
In Vitro Studies
5,21-30
(1976)
of Lymphocytes Kwashiorkor
from
Children
with
KURT SCHOPFER~ AND STEVEN D. DOUGLAS Nestle Foundation Research Center, Adiopodoume, Abidian, Ivory Coast, Departments of Medicine and Microbiology, University of Minnesota Medical Minneapolis, Minneapolis, Minnesota 5.5455
and School.
Received June 6, 1975 In vitro lymphocyte functions for children with kwashiorkor have been studied and compared to controls. Peripheral blood lymphocyte counts are similar to controls. Atypical mononuclear cells have been observed frequently in the peripheral blood smear. Electron microscopic observation revealed an increased number of plasmacytoid cells and occasionally lymphoblasts among isolated lymphocytes. In vitro response to phytohemagglutinin was decreased significantly, whereas pokeweed mitogen stimulation demonstrated higher response in 60% of the kwashiorkor patients studied and [3H]thymidine incorporation comparable to controls in 40%. The absolute number of rosette forming lymphocytes (T and B cell rosettes) was reduced in the kwashiorkor patients. Lymphocyte depletion of the thymus-dependent areas and reduction in the number of primary follicles and germinal centers were the main results of the histopathological examination of the thymolymphatic tissues. These findings suggest an acquired defect in cell-mediated immunity in kwashiorkor children.
INTRODUCTION
Increasing evidence for depression of the cell-mediated immune (CMI) response in children with protein-energy malnutrition (PEM) has accumulated since the relationship between malnutriton and infection has been recognized (l-3). Histopathologic findings, investigation of the in vitro lymphocyte function and in vivo testing, as well as clinical observations, suggest possible impairment of the thymus-dependent Q lymphocyte mediated immune reactions. Reports dealing with in vitro lymphocyte function have yielded conflicting results, which are probably attributable to the methodology employed or to a lack of clear definition of the syndrome involved. Therefore, the present study was undertaken to obtain further information about lymphocyte function of malnourished children. Accordingly, we have analyzed the rosette-forming ability, in vitro response to phytohemagglutinin (PHA-M) and pokeweed mitogen (PWM), and the electron microscopic features of lymphocytes from a group of children with kwashiorkor. PATIENTS AND METHODS
Twenty-seven children with a typical kwashiorkor syndrome admitted to the Pediatric Ward of the Centre Hospitalier de l’universite de Treichville, Abidjan, Ivory Coast, have been investigated and compared to a group of healthy control African children. Those with clinically overt infections were excluded from the study. Hematologic (hemoglobin, total leukocyte, and differential counts) and ’ Present address: Institute of Microbiology, Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
9008 St. Gallen, Switzerland.
22
SCHOPFER
AND
DOUGLAS
biochemical examinations (total protein, serum electrophoresis, cholinesterase activity) were performed by standard methods. Immunoglobulins (IgG, IgA, IgM) were measured by single radial immunodiffusion according to Mancini (4), using commercial immunodiffusion plates (Behringwerke, Marburg, Germany). Isolation of Lymphocytes The preparation of the lymphocytes was always performed within 2 hr after the blood samples were obtained. Lymphocytes were isolated from venous blood (containing nontoxic heparin, Upjohn, 20 units/ml) by the Ficoll-Hypaque density gradient method, as described by Boyum (5). The isolated cells were washed twice with phosphate buffered saline (PBS), pH 7.4, and suspended in the appropriate medium as indicated below. A differential count was performed and the lymphocyte number adjusted to the appropriate concentration. Rosette Formation Sheep red blood cell rosettes (SRBC-R) were prepared according to the method that we have previously described (6). In each preparation a minimum of 200 lymphocytes were counted in a hemocytometer and lymphocytes were designated rosette-positive if one or more SRBC’s adhered to the lymphocyte (“small” rosettes were defined as one or two adherent SRBC’s: “large” rosettes were lymphocytes with three or more attached SRBC’s.) Complement receptor lymphocytes (CRL) were assessed by the method of Michlmayr and Huber (7). The specimens were coded and identified after the percentage of rosette-forming cells was discerned. All preparations were examined by the same observer. Mitogen Stimulation Isolated lymphocytes were suspended in TC 199 containing 20% heat inactivated fetal calf serum (FCS) and adjusted with N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid (HEPES) to a pH of 7.4. PHA (Difco) or PWM (Gibco) was diluted in TC 199. Lymphocyte cultures were prepared that contained 3 x IO5 lymphocytes and 100 pg of PHA or PWM in a final volume of 0.3 ml per assay. Assays were performed in flat-bottom microculture plates (Falcon). Experiments were done in triplicate; samples without mitogen were always included. Cultures were incubated for 72 hr at 37°C. Eight hours before harvesting, 0.5 &i of [3H] thymidine (Amersham) were added. At the time of harvest, the incubation mixture was aspirated and filtered through a Millipore filter (1.2 pm pore size). The wells were washed repeatedly with PBS. The cell protein was then precipitated on the filter by successive washes with 10% followed by 5% trichloroacetic acid. The tilters were removed carefully, dried, and placed into a scintillation vial; 0.2 ml of 2 N KOH was added to dissolve the protein (8,9). After 30 min, scintillation fluid was added. The samples were counted in a liquid scintillation counter and the results were expressed as counts per minute. Electron Microscopy Isolated lymphocytes were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate containing sucrose. The samples were stored at 4°C. Cell preparations were postfixed in osmium tetroxide, dehydrated, and embedded in Epon. Specimens were stained en bloc with uranyl acetate. The samples were examined with a Siemens 102 electron microscope at an accelerating voltage of 80 V.
LYMPHOCYTES
IN
KWASHIORKOR
23
Histopathology Histopathologic examination of thymus, lymph node, spleen, and liver were performed for the single typical case in which it was possible to obtain autopsy material. RESULTS
Clinical and Laboratory Findings The children showed a typical kwashiorkor syndrome, as described earlier (10). The albumin levels (the main biochemical indicator of the degree of kwashiorkor) were always highly reduced (mean + SD): 1.85 ? 0.29 g/100 ml (n = 27) in comparison to controls: 3.88 + 0.45 g/100 ml (n = 23; p < 0.001). The total and differential leukocyte counts are summarized in Table 1. Higher neutrophil counts (p < 0.01) and a striking eosinopenia (p < 0.001) were encountered in the kwashiorkor children. There was no significant difference in the peripheral lymphocyte count. Atypical lymphocytes with a diminished nuclear/ cytoplasmic ration, increased basophilia, and cytoplasmic vacuolization were observed occasionally in the peripheral blood smear. Humoral Immunity Immunoglobulin levels are summarized in Table 2. No significant were observed between kwashiorkor children and controls.
differences
Rosette Formation SRBC-R. The total number of SRBC-R is lower in children with kwashiorkor @ < 0.05) than in the controls (Table 3). The percentage of rosette-forming lymphocytes, however, was similar in both groups. If lymphocytes to which three or more SRBC adhered were counted (“large rosettes”), then significantly fewer lymphocytes of kwashiorkor children formed rosettes QJ < 0.05). CRL. The absolute number of CRL was reduced for children with kwashiorkor (Table 3) @ < 0.05). There was no significant difference in the percentage of isolated lymphocytes binding to complement-coated SRBC. Mitogen Response PHA. The tritiated thymidine incorporation by lymphocytes from kwashiorkor children after 72 hr of incubation with PHA was diminished. The average values for the kwashiorkor patients were (mean + SD): 8917 + 6725 cpm (n = 15) compared to 24,149 f 8328 cpm (n = 28) for the controls. The difference is highly significant (p < 0.001) (Fig. 1). Cultures incubated without mitogens showed very low thymidine incorporation and there was no difference between kwashiorkor and control children. PWM. Determination of thymidine incorporation for lymphocytes incubated with PWM for 72 hr revealed two types of responses for cells from the kwashiorkor children. Lymphocytes from six children showed significantly increased thymidine incorporation (mean + SD: 12,516 + 2602) when compared to controls (mean f SD: 4613 + 2201; n = 9; p < 0.001). Cells from four of the 10 tested malnourished children showed values comparable to controls (Fig. 2). Electron Microscopy Pellets from Ficoll-Hypaque
preparations
revealed many typical small lympho-
33 ? 11 46 IL 13
2570 2 710 4380 k 2500 p < 0.01
-c 0.05
TABLE
p < 0.001
916 of:495 106 + 201
Absolute
IgG
1475 + 340” 1280 ? 130 ns.
(mg/lOO ml)
Total
62.3 t 7.1 57.2 ? 11.9 n.s.
.--Percentage
“Large”
w
2465 r 745 1147 2 531 < 0.05
65.3 t 10.9 32.1 t 10.4 c 0.05
“Small”
957 + 395 909 2 394 n.s.
Absolute
174 2 88 146 ? 39 n.s.
IgA
5*3 6~4
_--
17.0 r 6.6 75.1 t 5.3 i 0.05
Percentage
SRBC
tl.S.
131 ” 70 151 + 38
(mg/lOO ml)
340 _f 170 430 -t 300 ns.
Percentage
Monocytes Absolute
(mg/lOO ml)
LEVELS
Percentage
SRBC
SRBC-R (n =8)
Absolute
3
2
12 k 5 122
ROSETTE-FORMING ABILITY
TABLE
COUNTS
Percentage
Eosinophils
IMMUNOGLOBULIN
LYMPHOCVTE
1
LEUKOCYTE
TABLE BLOOD
’ All data mean t SD. * Student r- test; n.s., not significant.
3423 2 1043” 2055 or 747
Absolute
Ph
Controls (n = 14) Kwashiorkor (n = 18)
n All data mean -t SD. ’ Student t- test; n.s., not significant
Controls Kwashiorkor
P”
7576 I 2300 9066 + 4000 n.s.
Percentage
Neutrophils Absolute
” All data mean 2 SD. * Student r- test; n.s. = not significant.
Controls (28) Kwashiorkor (27) Significanceb
Total” Leukocytes
PERIPHERAL
4635 -t 1166 2727 ?z 1099 < 0.05
,4bsolute
14.6 ‘- 7.8 8.3 -c 4.2 n.s.
Percentage
_-_.-.--
50 t 9 47 2 11
Percentage
CRL (n =S)
3800 * 1600 4150 + 1900 n.s.
Absolute
Lymphocytes
w P
LYMPHOCYTES
IN KWASHIORKOR
25
-
Cuntrols
Kwashiorkor
FIG. 1. Comparison of the response to PHA of lymphocytes from children with kwashiorkor (n = 15) to controls (n = 28), cpm/3, lo5 lymphocytes. PHA Stimulation, 72 hr. (Individual cases; mean f SD)
cytes. There were frequent cytoplasmic lipid vacuoles, some cytoplasmic vacuolization, and occasional cells with increased cytoplasm and some increase in endoplasmic reticulum. About 10% of the cells seen had well-developed rough-surfaced endoplasmic reticulum in varying states of dilatation, features characteristic of plasmacytoid cells. However, plasmacytoid cells also were observed in some of the controls at a lower frequency. In addition, cells with large nucleoli and prominent euchromatin (lymphoblasts) were observed rarely in the kwashiorkor children (Fig. 3 and 4).
..
controls
Kwaohiorkar
FIG. 2. Comparison of the response to PWM of lymphocytes from children with kwashiorkor (n = 10) to controls (n = 9); cpm/3 x 105 lymphocytes. PWM Stimulation, 72 hr.
26
SCHOPFER
AND
DOUGLAS
FIG. 3. Electron micrograph of Ficoll-Hypaque mononuclear cell pellet from a child wi ith kwashiorkor. Note the lymphoblast with prominent nucleoli; several of the small lymphocytes also coIntain nucleoli. x 5100.
FIG. 4. Electron micrograph of plasmacytoid cell with dilated rough-surfaced re:ticulum from peripheral blood of kwashiorkor patient. x 14,000.
endoplasm lie
LYMPHOCYTES
IN
KWASHIORKOR
27
Histopathology Pertinent histopathologic findings include: marked thymic atrophy with difftculty in identification of typical Hassall’s corpuscles; depletion of the paracortical areas of lymph nodes and reduction in germinal centers, and primary follicles; depletion of the periarteriolar areas of the spleen; diffusely distributed lymphoblastoid cells with few discernible typical plasma cells and frequent occurrence of histiocytes in the sinusoidal areas of thymus, lymph nodes, spleen, and liver (Fig. 5 and 6).
FIG. 5. Spleen from autopsy of kwashiorkor tion. X 80.
patient. Periarteriolar
areas show lymphocyte deple-
FIG. 6. Thymic lobule from kwashiorkor patient. The lymphocyte depletion and paucity of readily identifiable Hassalls’ corpuscles are noted. x 80.
DISCUSSION
The present investigation of children with kwashiorkor showed normal peripheral blood lymphocyte counts, occasional atypical lymphocytes, decreased numbers of SRBC rosettes (T cells) and reduced numbers of CRL (B cells) when compared to controls. The response to PHA was diminished in the kwashiorkor children; tritiated thymidine incorporation with PWM incubation revealed two
28
SCHOPFER
AND
DOUGLAS
types of response (values comparable to controls in four children, significantly higher response in six children). About 10% of the isolated mononuclear cells showed plasmacytoid features by electron microscopy; blastoid cells were found rarely. The postmortem morphologic features of the lymphoreticular system included: severe thymic atrophy with absence of identifiable typical Hassall’s corpuscles, depletion of lymphocytes in the thymus-dependent areas of the lymph nodes and spleen, as well as decreased numbers of primary follicles and lack of germinal centers. Rather striking features were the presence of diffusely distributed lymphoblastoid cells and a prominent infiltration of the sinusoids of the lymphoreticular system with histiocytes. Studies by other investigators have revealed abnormalities ofin vitro lymphocyte function in malnourished children. Chandra (1 I) and Ferguson et ~1. (12) found decreased percentages of SRBC-R forming lymphocytes, but did not report absolute numbers. Reduced tritiated thymidine incorporation by lymphocytes following incubation with phytohemagglutinin has been demonstrated (13,14) and morphologic studies of blast transformation also showed diminished numbers of transformed cells with PHA (15-17). Our histopathologic findings are consistent with those reported by others (18-20). These observations may reflect a reduction in the circulating T cell pool size, changes in lymphocyte recirculation, and/or impaired differentiation or membrane abnormalities. Diminished PHA response and decreased rosette-forming ability are probably evidence for an abnormality of the circulating T cell population. The significance of “small” and “large” rosettes is unknown, yet it is possible that alterations in membrane properties are related to the observations of reduced numbers of “large” rosettes. Impairment of delayed hypersensitivity in malnourished children has been demonstrated in Go using PPD (21), DNCB ( 15, 22), Candida antigen (2), and keyhole limpet hemocyanin (KLH) (2). The increased incidence of infections and the unusual course of some of these infections (e.g., high incidence of measles giant cell pneumonia) (19,23) are further clinical evidence for abnormal CMI. The in vitro lymphocyte studies reported here and by others correlate with the abnormal CM1 suggested by clinical observations of these patients. Preliminary data suggest significantly lower absolute numbers of CRL (a B cell marker) in kwashiorkor children; however, the percentage ofCRL was not different compared to controls. The number of cases investigated is too small to draw definitive conclusions and other B cell markers must be studied. The presence of plasmacytoid cells in the peripheral blood is an unusual finding and has been reported in viral infections (24, 25) following thermal injury (26), in serum sickness (27), in Waldenstrom’s macroglobulinemia(28), in sarcoidosis (29), and after ingestion of berries or roots of Phytolucca americana (pokeweed) (30). The depletion of the B cell areas in lymph nodes and spleen, the observation of plasmacytoid cells in the peripheral blood, and the elevated response of lymphocytes from some patients to PWM could reflect the release of differentiated B cells into the circulation in kwashiorkor. The precise correlation between the morphology of lymphocyte-plasma cell development, mitogenic responsiveness, and surface markers is not yet known in clinical states.
LYMPHOCYTES
IN
KWASHIORKOR
29
Immunoglobulin levels are not different between kwashiorkor children and controls in our investigation. Reports dealing with the determination of immunoglobulin levels have shown conflicting results. Increased, normal, and reduced levels (3 I-35) have been found. The presence of infections, as well as the type and degree of malnutrition, must be considered in all evaluations. Our findings and those of others suggest that in a substantial proportion of kwashiorkor children, features of impaired CM1 are present, comparable to other clinical conditions characterized by defective CM1 function; yet the B cell dependent system in kwashiorkor children appears to be intact. Nevertheless, a number of unusual features remain to be unraveled. Immunodeficiency syndromes, either genetic or acquired, generally are associated either with peripheral blood lymphopenia (diminished numbers of B and/or T lymphocytes) and/or low immunoglobulin levels (36). Neither of these findings could be found consistently in kwashiorkor children. The lymphocyte depletion of the thymolymphatic tissues in kwashiorkor children is striking and it would be of importance of ascertain how normal, or only slightly reduced lymphocyte counts are maintained. Lymphocytopenia could be further expected by the hypercortisolemia (38, 39), which is always encountered in kwashiorkor children. Furthermore, the normal immunoglobulin levels remain to be explained. Reduction of the bone marrow-dependent B cell areas, few primary follicles, and germinal centers have been demonstrated in histopathologic examination and yet there is no reduction in immunoglobulins found. There is an increased preponderance of plasmacytoid cells in the peripheral blood, as well as an increased response to pokeweed mitogen, but this does not necessarily imply increased immunoglobulin synthesis, although hypergammaglobulinemia occasionally occurs in children with kwashiorkor. The situation is further complicated in kwashiorkor due to the hepatic steatosis and the severe liver disease with the possibility of impaired degradation of intestinal antigens. The role of the portal system in antigen presentation and antigen clearing has been investigated in patients with cirrhosis, where hypergammaglobulinemia and plasmacytoid cells are frequently observed (39,40). The role of this system in kwashiorkor should be further investigated. ACKNOWLEDGMENTS This work has been supported by the Nestle Foundation for the Study of the Problems ofNutrition in the World, the World Health Organization, and USPHSGrant AI-12478-01 from the National Institutes of Health. We thank Professors S. Badoual and N. Esso of the CHU Treichville for permission to study these patients. We are grateful to Sister Christine for her help. We thank Mr. J. F. Bois, Chief, Laboratory of Radioisotopes, ORSTOM, Abidjan, Ivory Coast for making scintillation counters available. We also appreciate the dedication and excellent technical assistance of Miss C. G. Mayer and Mrs. D. Schopfer.
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