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T cell subsets in acute rheumatic fever, rheumatic heart disease and acute glomerulonephritis cases
Immunology Letters, 15 (1987) 217-219 Elsevier IML 00901
T cell subsets in acute rheumatic fever, rheumatic heart disease and acute glomerulonephritis cases P r a v i n K. B h a t n a g a r , Reba N i j h a w a n a n d Kunti P r a k a s h WHO Collaborating Centre for Reference and Training in Streptococcal Diseases, Department of Microbiology, Lady Hardinge Medical College and Smt. Sucheta Kriplani Hospital, New Delhi, India (Received 24 November 1986) (Revision received 13 April 1987) (Accepted 14 April 1987)
1. Summary
3. Materials and Methods
T cell subsets were studied in 45 patients, 15 each of acute rheumatic fever, rheumatic heart diseases, and acute glomerulonephritis and compared with 10 normal control in the same age group. A significant depression in the OKT1 and OKT4 +ve cells and insignificant increase inthe OKT8 +ve ceils was noted in all these groups of cases.
3.1. Patients Forty-five patients (5-15 year age group), 15 each of ARF, R H D and AGN, diagnosed clinically and on the basis of bacteriological and serological findings constituted the subject of the study. Only those patients who did not receive any prior corticosteroids were included in the study. Ten ageand sex-matched children with no bacteriological and serological evidence of streptococcal infections were included as controls.
2. Introduction
Antecedent group A beta-haemolytic streptococcal infection (BHS) is clearly somehow related to the acute rheumatic fever process [1, 2]. The low T cell counts in peripheral blood of acute rheumatic fever (ARF), rheumatic heart disease (RHD) and acute poststreptococcal glomerulonephritis (AGN) patients have long been reported [ 3 - 6 ] . There are scanty and conflicting reports on T cell subsets in ARF and RHD patients [ 6 - 8 ] and to our knowledge none in AGN cases. In the present study we have estimated the T cell subset population in patients suffering from ARF, R H D and AGN. Key words: T cell subset; Acute rheumatic fever; Rheumatic heart disease; Acute glomerulonephritis
Correspondence to: Dr. P. K. Bhatnagar, WHO Collaborating Centre for Reference and Training in Streptococcal Diseases, Department of Microbiology, Lady Hardinge Medical College and Smt. Sucheta Kriplani Hospital, New Delhi-110001, India.
3.2. Cell separation Five ml of heparinised blood (10 IU/ml heparin) was drawn and their lymphocytes were separated by the method described previously [4]. The pan T cell, helper and suppressor cells were identified using OKT1, OKT4 and OKT8 monoclonal antibodies (Dakopatt), respectivelY, by indirect immunofluorescence [7]. The staining was carried out on poly-l-lysine (average MW 70,000, P2636 sigma) coated VDRL slides [9]. 3.3. Coating of the slides Ten to twenty /A of 100 /~g/ml poly-l-lysine in phosphate buffered saline (PBS) were placed over each well of the slide and kept at room temperature for 25 min. The slides were then rinsed with distilled water and air dried. 3.4. Characterization of cells The coated slides were kept in cold prior to use.
0165-2478 / 87 / $ 3.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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Twenty /A of lymphocytes (106) were placed over each well for 45 min at 4°C. The cells were then washed three times with cold PBS and treated with 10 /A of primary antibody (OKT1, OKT4 and OKT8) for 45 min at 4 °C, washed three times with cold PBS and incubated with secondary antibody (antimouse FITC-conjugated antibody F(ab)2 fraction) for 45 min at 4°C. Cells were washed and mounted with 1007oglycerol phosphate buffered saline and examined under fluorescent microscope. Two-hundred ceils were counted to calculate the percentage of fluorescing cells.
5. Discussion
The present study further supported our earlier observation that one of the major changes during the active stage of A R F / R H D and AGN cases was the depression in the T cells [4, 5]. Our findings are in accordance with earlier reports [3, 6]. Our findings further showed a significant decrease in both percentage and absolute number of OKT4 + ve'cells in A R F / R H D and AGN cases. Similar findings were observed in ARF [7, 8] and R H D [6] patients. The depression in the OKT1 +ve cells as a whole and OKT4 + ve cells may be due to the fact that T cell population is disproportionally retained at the tissue site of active inflammation [7]. This possibility was further supported by Raizada et al. [12] who showed the predominance of OKT4 + ve cells and only few OKT8 +ve cells in the mononuclear infiltrates (70-80°70 T cells) occurring in the cardiac valvular tissue of R H D patients. Williams et al. [7] reported a decrease in OKT8 + ve cells in ARF patients. We have found a tendency towards increased OKT8 + ve ceils though was not statistically significant. Ganguly et al. [6] using T/~ and T r assay observed a significant increase in the percentage of suppressor/cytotoxic cells in active RHD patients and suggested these may have a pathogenic role in patients with rheumatic activity. The significance of the findings o f significantly lower OKT1 +ve cells, OKT4 +ve cells and increased OKT8 +ve (only in percentage) cells in
3.5. Statistical analysis Data obtained were statistically analysed using Student's t-test.
4. Results
The values of pan T cells as identified by OKT1 as well as putative helper (OKT4) and suppressor (OKT8) T cells estimated in ARF, R H D and AGN cases are shown in Table 1. It was observed that OKT1 + ve cells were significantly low in all these groups of cases (Table 1). A significant decrease in the proportion and absolute number of OKT4 +ve T cells was recorded in ARF (P<0.05), RHD (P<0.05) and AGN (P<0.01) cases. OKT8 +ve cells on the other hand, appeared to be slightly on the higher side in terms of percentage; however, this also did not reach statistical significance.
Table 1 T-cells and T-cell subsets in children suffering from A R F / R H D / A G N . Subject
No. of cases
Controls ARF
10 15
RHD
15
AGN
15
Pan T cells (OKT1 + ve)
OKT4 + ve cells
OKT8 + ve cells
%
Abs. counts/3 mm
%
Abs. counts/3 mm
%
Abs. counts/3 mm
75.8 +2.8 67.86±2.84 (<0.001) 70.0 +2.79 (<0.01) 65.4 ±3.62
2547+_503 1982±384 (<0.01) 2054±460 (<0.05) 1871+513
54 +3.5 41.4+4.3
1468 ± 432 1130_+243
(
(<0.05)
40.8 _+4.4 42.8 + 4.6 (NS) 45.1 + 5.2 (NS) 44.2±7
(<0.0Ol)
(<0.01)
(<0.05)
1110+372 1116_+157 (NS) 1062_+ 191 (NS) 897 + 290 (NS)
P values in parentheses. NS = not significant.
218
44.8+6
1082 + 247
(<0.05)
(< 0.05)
47.4±6.3
948 ___398 (<0.01)
(NS)
A G N cases is n o t clear. H o w e v e r , it is k n o w n [13] that lymphocytes and monocytes infiltrate g l o m e r u l o n e p h r i t i c kidney. F u r t h e r s t u d y o f t h e h i s t o l o g i c a l p i c t u r e o f t h e g l o m e r u l o n e p h r i t i c kidney m a y s u g g e s t t h e i r role in c a u s i n g r e n a l d a m a g e , if at all.
Acknowledgement T h e t e c h n i c a l a s s i s t a n c e o f Mr. A z h a r K h a n is gratefully acknowledged.
References [1] Kantanzaro, E J., Stetson, C. A., Morris, A. J., Chemovitz, R., Rammelkamp, C. H. Jr., Stolzer, B. L. and Perry, W. R. (1954) Am. J. Med. 17, 749. [2] McCarty, M. (1956) Circulation 14, 1138. [3] Lueker, R. D., Abdin, Z. H. and Williams, R. C. Jr. (1975) J. Clin. Invest. 55, 975.
[4] Prakash, K., Bhatnagar, P. K. and Sharma, K. B. (1983) Indian J. Med. Res. 78, 1. [5] Prakash, K. and Bhatnagar, P. K. (1984) Indian J. Med. Res. 80, 284. [6] Ganguly, N. K., Anand, I. S., Khanna, A. K., Kohli, E S. and Wahi, P. L. (1982) Indian J. Med. Res. 76, 854. [7] Williams, R. C., Raizada, V., Prakash, K., Van de Rijin, I., Zabriskie, J. B., Stobo, J. D. and Sharma, K. B. (1983) J. Clin. Immunol. 2, 166. [8] Garraud, O., Ribiere, O. and Bach, J. E (1986) Immunol. Lett. 13, 75. [9] Van Voorhis, W. C., Valinsky, J., Hoffman, E., Luban, J., Hair, L. S. and Steinman, R. M. (1983) J. Exp. Med. 158, 174. [10] Reinherz, E. L., Kung, P. C., Goldstein, G. and Schlossman, S. F. (1979) J. Immunol. 123, 2894. [11] Reinherz, E. L., Kung, P. C., Goldstein, G. and Schlossman, S. E (1980) J. Immunol. 124, 1301. [12] Raizada, V., Williams, R. C., Chopra, P., Prakash, K., Sharma, K. B., Cherian, K. M., Pandey, S., Arora, R., Nigam, M., Zabriskie, J. B. and Husby, G. (1983) Am. J. Med. 74, 90. [13] Rocklin, R. E., Lewis, E. J. and David, J. R. (1970) N. Engl. J. Med. 283, 493.
219
T cell subsets in acute rheumatic fever, rheumatic heart disease and acute glomerulonephritis cases P r a v i n K. B h a t n a g a r , Reba N i j h a w a n a n d Kunti P r a k a s h WHO Collaborating Centre for Reference and Training in Streptococcal Diseases, Department of Microbiology, Lady Hardinge Medical College and Smt. Sucheta Kriplani Hospital, New Delhi, India (Received 24 November 1986) (Revision received 13 April 1987) (Accepted 14 April 1987)
1. Summary
3. Materials and Methods
T cell subsets were studied in 45 patients, 15 each of acute rheumatic fever, rheumatic heart diseases, and acute glomerulonephritis and compared with 10 normal control in the same age group. A significant depression in the OKT1 and OKT4 +ve cells and insignificant increase inthe OKT8 +ve ceils was noted in all these groups of cases.
3.1. Patients Forty-five patients (5-15 year age group), 15 each of ARF, R H D and AGN, diagnosed clinically and on the basis of bacteriological and serological findings constituted the subject of the study. Only those patients who did not receive any prior corticosteroids were included in the study. Ten ageand sex-matched children with no bacteriological and serological evidence of streptococcal infections were included as controls.
2. Introduction
Antecedent group A beta-haemolytic streptococcal infection (BHS) is clearly somehow related to the acute rheumatic fever process [1, 2]. The low T cell counts in peripheral blood of acute rheumatic fever (ARF), rheumatic heart disease (RHD) and acute poststreptococcal glomerulonephritis (AGN) patients have long been reported [ 3 - 6 ] . There are scanty and conflicting reports on T cell subsets in ARF and RHD patients [ 6 - 8 ] and to our knowledge none in AGN cases. In the present study we have estimated the T cell subset population in patients suffering from ARF, R H D and AGN. Key words: T cell subset; Acute rheumatic fever; Rheumatic heart disease; Acute glomerulonephritis
Correspondence to: Dr. P. K. Bhatnagar, WHO Collaborating Centre for Reference and Training in Streptococcal Diseases, Department of Microbiology, Lady Hardinge Medical College and Smt. Sucheta Kriplani Hospital, New Delhi-110001, India.
3.2. Cell separation Five ml of heparinised blood (10 IU/ml heparin) was drawn and their lymphocytes were separated by the method described previously [4]. The pan T cell, helper and suppressor cells were identified using OKT1, OKT4 and OKT8 monoclonal antibodies (Dakopatt), respectivelY, by indirect immunofluorescence [7]. The staining was carried out on poly-l-lysine (average MW 70,000, P2636 sigma) coated VDRL slides [9]. 3.3. Coating of the slides Ten to twenty /A of 100 /~g/ml poly-l-lysine in phosphate buffered saline (PBS) were placed over each well of the slide and kept at room temperature for 25 min. The slides were then rinsed with distilled water and air dried. 3.4. Characterization of cells The coated slides were kept in cold prior to use.
0165-2478 / 87 / $ 3.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
217
Twenty /A of lymphocytes (106) were placed over each well for 45 min at 4°C. The cells were then washed three times with cold PBS and treated with 10 /A of primary antibody (OKT1, OKT4 and OKT8) for 45 min at 4 °C, washed three times with cold PBS and incubated with secondary antibody (antimouse FITC-conjugated antibody F(ab)2 fraction) for 45 min at 4°C. Cells were washed and mounted with 1007oglycerol phosphate buffered saline and examined under fluorescent microscope. Two-hundred ceils were counted to calculate the percentage of fluorescing cells.
5. Discussion
The present study further supported our earlier observation that one of the major changes during the active stage of A R F / R H D and AGN cases was the depression in the T cells [4, 5]. Our findings are in accordance with earlier reports [3, 6]. Our findings further showed a significant decrease in both percentage and absolute number of OKT4 + ve'cells in A R F / R H D and AGN cases. Similar findings were observed in ARF [7, 8] and R H D [6] patients. The depression in the OKT1 +ve cells as a whole and OKT4 + ve cells may be due to the fact that T cell population is disproportionally retained at the tissue site of active inflammation [7]. This possibility was further supported by Raizada et al. [12] who showed the predominance of OKT4 + ve cells and only few OKT8 +ve cells in the mononuclear infiltrates (70-80°70 T cells) occurring in the cardiac valvular tissue of R H D patients. Williams et al. [7] reported a decrease in OKT8 + ve cells in ARF patients. We have found a tendency towards increased OKT8 + ve ceils though was not statistically significant. Ganguly et al. [6] using T/~ and T r assay observed a significant increase in the percentage of suppressor/cytotoxic cells in active RHD patients and suggested these may have a pathogenic role in patients with rheumatic activity. The significance of the findings o f significantly lower OKT1 +ve cells, OKT4 +ve cells and increased OKT8 +ve (only in percentage) cells in
3.5. Statistical analysis Data obtained were statistically analysed using Student's t-test.
4. Results
The values of pan T cells as identified by OKT1 as well as putative helper (OKT4) and suppressor (OKT8) T cells estimated in ARF, R H D and AGN cases are shown in Table 1. It was observed that OKT1 + ve cells were significantly low in all these groups of cases (Table 1). A significant decrease in the proportion and absolute number of OKT4 +ve T cells was recorded in ARF (P<0.05), RHD (P<0.05) and AGN (P<0.01) cases. OKT8 +ve cells on the other hand, appeared to be slightly on the higher side in terms of percentage; however, this also did not reach statistical significance.
Table 1 T-cells and T-cell subsets in children suffering from A R F / R H D / A G N . Subject
No. of cases
Controls ARF
10 15
RHD
15
AGN
15
Pan T cells (OKT1 + ve)
OKT4 + ve cells
OKT8 + ve cells
%
Abs. counts/3 mm
%
Abs. counts/3 mm
%
Abs. counts/3 mm
75.8 +2.8 67.86±2.84 (<0.001) 70.0 +2.79 (<0.01) 65.4 ±3.62
2547+_503 1982±384 (<0.01) 2054±460 (<0.05) 1871+513
54 +3.5 41.4+4.3
1468 ± 432 1130_+243
(
(<0.05)
40.8 _+4.4 42.8 + 4.6 (NS) 45.1 + 5.2 (NS) 44.2±7
(<0.0Ol)
(<0.01)
(<0.05)
1110+372 1116_+157 (NS) 1062_+ 191 (NS) 897 + 290 (NS)
P values in parentheses. NS = not significant.
218
44.8+6
1082 + 247
(<0.05)
(< 0.05)
47.4±6.3
948 ___398 (<0.01)
(NS)
A G N cases is n o t clear. H o w e v e r , it is k n o w n [13] that lymphocytes and monocytes infiltrate g l o m e r u l o n e p h r i t i c kidney. F u r t h e r s t u d y o f t h e h i s t o l o g i c a l p i c t u r e o f t h e g l o m e r u l o n e p h r i t i c kidney m a y s u g g e s t t h e i r role in c a u s i n g r e n a l d a m a g e , if at all.
Acknowledgement T h e t e c h n i c a l a s s i s t a n c e o f Mr. A z h a r K h a n is gratefully acknowledged.
References [1] Kantanzaro, E J., Stetson, C. A., Morris, A. J., Chemovitz, R., Rammelkamp, C. H. Jr., Stolzer, B. L. and Perry, W. R. (1954) Am. J. Med. 17, 749. [2] McCarty, M. (1956) Circulation 14, 1138. [3] Lueker, R. D., Abdin, Z. H. and Williams, R. C. Jr. (1975) J. Clin. Invest. 55, 975.
[4] Prakash, K., Bhatnagar, P. K. and Sharma, K. B. (1983) Indian J. Med. Res. 78, 1. [5] Prakash, K. and Bhatnagar, P. K. (1984) Indian J. Med. Res. 80, 284. [6] Ganguly, N. K., Anand, I. S., Khanna, A. K., Kohli, E S. and Wahi, P. L. (1982) Indian J. Med. Res. 76, 854. [7] Williams, R. C., Raizada, V., Prakash, K., Van de Rijin, I., Zabriskie, J. B., Stobo, J. D. and Sharma, K. B. (1983) J. Clin. Immunol. 2, 166. [8] Garraud, O., Ribiere, O. and Bach, J. E (1986) Immunol. Lett. 13, 75. [9] Van Voorhis, W. C., Valinsky, J., Hoffman, E., Luban, J., Hair, L. S. and Steinman, R. M. (1983) J. Exp. Med. 158, 174. [10] Reinherz, E. L., Kung, P. C., Goldstein, G. and Schlossman, S. F. (1979) J. Immunol. 123, 2894. [11] Reinherz, E. L., Kung, P. C., Goldstein, G. and Schlossman, S. E (1980) J. Immunol. 124, 1301. [12] Raizada, V., Williams, R. C., Chopra, P., Prakash, K., Sharma, K. B., Cherian, K. M., Pandey, S., Arora, R., Nigam, M., Zabriskie, J. B. and Husby, G. (1983) Am. J. Med. 74, 90. [13] Rocklin, R. E., Lewis, E. J. and David, J. R. (1970) N. Engl. J. Med. 283, 493.
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