Cell mediated immunity was assessed in 30

children with acute post streptococcal glomerulonephritis

(APSGN) in parallel with 20 normal children

and 15 children without nephritis who showed evidence

of skin-sore ß-Hemolytic streptococcal

infection. Delayed cutaneous hypersensitivity to

2,4, dinitrochloro benzene (DNCB) was similar in

the three groups. There were no significant differences

in the proportion of early and total T-rosettes.

Lymphocyte transformation response to phytohemagglutinin-

P (PHA), purified protein derivative

(PPD) and BCG was similar in them, indirect leucocyte

migration inhibition response to PPD, streptokinase

streptodornase (SK-SD), and group A ß-Hemolytic

T12 streptococcal antigens were not significantly

different in patients when compared to normal controls

and streptococcal infection controls. Cell mediated

immunity was normal in APSGN in children by all

the parameters studied

Moses, S G P

Narayanan, P R

Rajajee, S

Sundaravalli, N

NIRT Institutional Repository

VOLUME 21 – AUGUST 1984CELL MEDIATED IMMUNITYIN POST-STREPTOCOCCALGLOMERULONEPHRITISS. RajajeeP.R. NarayananS.G.P. MosesN. SundaravalliABSTRACTCell mediated immunity was assessed in 30children with acute post streptococcal glomerulo-nephritis (APSGN) in parallel with 20 normal childrenand 15 children without nephritis who showed evi-dence of skin-sore ß-Hemolytic streptococcalinfection. Delayed cutaneous hypersensitivity to2,4, dinitrochloro benzene (DNCB) was similar inthe three groups.There were no significant diffe-rences in the proportion of early and totalT-rosettes.Lymphocyte transformation response to phyto-hemagglutinin-P (PHA), purified protein derivative(PPD) and BCG was similar in them, indirect leuco-cyte migration inhibition response to PPD, strepto-kinase streptodornase (SK-SD), and group A ß-Hemo-lytic T12 streptococcal antigens were not significantlydifferent in patientswhen compared to normal cont-rols and streptococcal infection controls. Cell mediatedimmunity was normal in APSGN in children by allthe parameters studied.Key words: Cell mediated immunity, Acute post-streptococcal glomerulonephritis.The prevalence of APSGN has declinedin the developed countries but it remainsrelatively common in tropical developingcountries.Epidemics of APSGN follow-ing skin infection such as Impetigo orinfected scabies have been reported fromseveral parts of the world(1-3).At theInstitute of Child Health, Madras, anaverage of 432 cases of APSGN are admit-ted per annum with an average of 35.8per month, majority of the cases followedinfected scabies or impetigo. The factorsresponsible for the high incidence are poorhygiene and over-crowding which predis-pose to recurrent scabies and impetigo.The role of undernutrition in predisposingto APSGN has to be assessed becauseof the depressive effect of undernutritionon cell mediated immunity(4,5).The important role played by thehumoral immune system in the patho-genesis of glomerulonephritis is well knownwhile the involvement of cell mediatedImmunity-is doubtful.There is very little reported data oncell mediated immunity in APSGN inchildren.The present study was undertaken toassess the cell mediated immunity (CMI)during the acute phase of APSGN, andthis was compared to the CMI in skininfection (contact) controls and normalc ntrols.Material and MethodsThe study consisted of 3 groups ofchildren.(A) APSGN (30 children) during theacute phase.The following criteria were taken toFrom the Department of Pediatricsand Medicine,classify them:Madras Medical College and Department of(1) Acute onset of oliguria and edemaImmunology, Tuberculosis Research Centre,(2) Proteinuria and microscopic or grossMadras. hematuria615RAJAJEE ET AL. CELL MEDIATED IMMUNITY IN GLOMERULONEPHRITIS(3) Transient rise of blood pressure.(4) Other clinical features such as pul-monary edema,seizures or renalfailure.(5) No past history of renal disease.(6) Evidence of previous streptococcalinfection such as raised Anti-D NaseB levels and/or positive culture.(7) Low C3 levels.(B) Skin infection (contact) controls (15children) consisted of siblings ofpatients with skin sepsis but normalurinary findings, raised anti-D NaseB levels and/or positive culture andnormal C3 levels.(C) Norma1 children (20 children) werechildren of employees attending amedical centre, with no evidence ofskin infection, normal urinary find-ings, anti-D Nase B levels and C3levels.The nutritional status of the childrenwas assessed after discharge, duringfollow up and graded according to ICMRreference standards.Delayed cutaneous hypersensitivity toDNCB, (Koch-light Laboratories, U.K.)was tested by the method of Catalonaet al. (6) but with the lower sensitising doseof 500 µg/ml because at the higher dose of1000 µg/ml severe ulceration occurred(7).Lymphocyte separationSamples of peripheral blood weredrawn from the children. Lymphocyteswere isolated by Ficoll Hypaque densitygradient centrifugation. The cells at theinterface were collected, washed thrice inHank’s Balanced salt solution, the viabi-lity was approximately 95 % cells as revea-led by the Trypan blue exclusion test.Cell surface markersT-cells were enumerated in two sepa-616rate determinations using the rapid rosettetechnique(8) as well as total sheep cellrosettes incubated at 4°C and read after12 to 18 hours incubation(9).Lymphocyte transformationThe lymphocytes were cultured induplicates in 1 ml of RPMI 1640 supple-mented with penicillin (100 µg/ml), streptomycin 100 µg/ml glutamine (300 µg/ml)and 0.1 ml autologous plasma in 24 welltis ue culture plates (Laxbro) at a con-ce tration of 1 x 106 cells per ml. Thecells were stimulated with1 µg/mlphytohemagglutin P (PHA-P-WellcomeBurroughs), 50 µg/ml purified proteinderivative (PPD-preservative free CentralVeterinary Lab., U.K.) and 50 µg/ml BCG(BCG Laboratories, Madras). Cultureswere incubated at 37°C in an atmosphereof 5% CO2 for 96 hours for PHA and 144hours for PPD and BCG.The proliferative response was mea-sured by adding 10 uci of 3 H Thymidine(Sp Act 13000 MCi/Mol Babha AtomicResearch Centre, Bombay) 16 hours be-fore harvesting. At the time of harvest,0.2 ml of lymphocyte cell suspension wastransferred from each well of 24 wellplates into 96 well plates in triplicates,subsequently harvesting was done withMASH II (Microbiological associatesUSA) and deposited on (Whatman) fibreglass paper. Paper discs were then trans-ferred to biovials containing 1 ml ofscintillation fluid and counted in a scintil-lation counter. Stimulation Index wascalculated as follows:CPM in stimulated culturesS.I. =CPM in control culturesIndirect leucocyte migration inhibitiont st.INDIAN PEDIATRICS VOLUME 21 – AUGUST 1984Streptococcal antigensPure cultures of Group A ß-hemolyticstreptococcus T12which was isolated froma nephritic patient was used.The bacterial suspension was killedby Beating at 70°C for 1 hour in a waterbath, different wet weight concentrationswere used, optimal concentration wasfound to be 1 mg/ml.LymphokinesPurified lymphocytes obtained fromperipheral blood as described earlier weresuspended in RPMI 1640 with penicillin100 µg/ml streptomycin 100 µg/ml,glutamine 300 µg/ml with 10% sterilehorse serum at a final concentration of1 x 106 cells/ml. The cells were stimu-lated with 50 µg/ml PPD, 200 units/mlSK-SD and 1 mg/ml streptococcal bacil-lary suspension with appropriate controls.The cultures were incubated at 37°Cin an atmosphere of 5 % CO2 for 96 hours.After centrifugation, the supernatant wasseparated.Peritoneal cells were collected fromhealthy guinea pigs after stimulation withliquid paraffin. The peritoneal cells wereallowed to migrate in 12 well LMI plates(Laxbro) surrounded by lymphokinesobtained in the presence of antigens andcontrols. The plates were incubated at37°C for 18 hours, migration patternswere projected at fixed magnification andtraced. The projected areas were mea-sured by cutting out and weighing. Theassay was set up in triplicates.Migration Index (MI) =Area of migration in the presenceof antigensArea of migration in controlsMI below 0.8 was taken as indicationof positive reactors.ResultsTable I indicates the nutritional statusof the patients and skin infection controls.Majority are normal weight for age or hadmild undernutrition and the children inboth the groups were in the same nutri-tional range.Delayed cutaneous hypersensitivityto DNCB was positive in all the patientsas well as skin infection and normal con-trols ranging from 4+ to 1+ (Table II).The proportion of early T-rosetteswas 22.4, 22.2 and 20.2% in patients,normal controls and skin infection con-trols respectively. There was also nosignificant difference in the proportion oftotal T-rosettes which was 58.6% inpatients, 59.25% in normal controls and57.4% in skin infection controls. This isindicated in Table III.The mean values of the mitogenicresponse to PHA of patients, skin infec-tion (contact) controls and normal con-trols are shown in Table IV. T-cell functionas measured by the response to PHA inautologous plasma was normal in patientsas compared to the response in skininfection controls and normal controls.The lymphocyte response of patientsto PPD and BCG was not significantlydifferent from that of skin infection andnormal controls (Table IV).Similarly, in the indirect leucocytemigration inhibition test the mean valuesfor migration index in response to PPDwas not different in patients when com-pared to controls (Table V).The responsiveness of the lymphocytesto SK-SD was similar in patients, skininfection controls and normal controls(Table V).617RAJAJEE ET AL. CELL MEDIATED IMMUNITY IN GLOMERULONEPHRITISTABLE I – Nutritional status of children with APSGNGrading of nutritionSubjects No.Normal 1 2 31. Acutenephritis 135 72 50 13 –2. Skin infection (controls)32 14 14 4 –3. Normal (controls) 20 12 6Classified using ICMR Reference StandardsUpto 80% of Standard for weight-Normal70–80% -Mild undernutrition60–70% -Moderate undernutritionBelow 60% -Severe undernutrition2 –TABLE II – Delayed cutaneous hypersensitivity to DNCB in APSGN sensitisation dose 500 µg/0.1 mlchallenge dose 50 µg/0.1 mlGroup No. of subjects4+DNCB positive3+ 2+DNCB1+ negativeAcute Nephritis 60 20 30 6 4 NilNormal (control) 50 17 31 2 – NilSkin infection (control) 15 5 9 1 – NilTABLE III – Comparison of the peripheral blood early and total lymphocytes in ASPGN and controlsGroup No. % %Early T-rosettes Total T-rosettesAcute nephritisNormal (controls)30 22.4 ± 6.7 58.6 ± 8.10(8-36) (44-72)20 22.2 ± 4.5 59.25 ± 6.2(13-30) (46-70)Skin infection (controls) 5 20.2 ± 4.14 57.4 ± 6.6(16-27) (47-64)618INDIAN PEDIATRICS VOLUME 21 – AUGUST 1984TABLE IV – Lymphocyte transformation response to PHA, PPD and BCG in APSGN and controlsGroupPHAStimulation index mean and SDPPD BCGAcute nephritisNormal (controls)Skin infection (controls)*Number of subjects.43.9 ± 31.3 4.6 ± 7.3 7.6 ± 16(26)* (26) (23)41.7 ± 26.1 5.7 ± 11.8 2.8 ± 1.3(15) (13) (11)48.6 ± 7.3 2.10 ± 2.1 4.01 ± 1.5(5) (5) (5)TABLE V – Indirect LMI response to streptococcol antigens SK/SD and PPD in APSGN and controlsGroupAcute nephritisNormal (controls)Skin infection (controls)Migration index mean and SDStreptococcal antigens SK-SD0.76 ± 0.6 0.68 ± 0.5(32)* (15)0.75 ± 0.6 0.65 ± 0.3(15) (12)0.93 ± 0.4 0.69 ± 0.5(10) (10)PPD0.76 ± 0.7(15)0.45 ±  0.3(14)–*Number of subjects.Response to the streptococcal bacillarysuspension showed no significant differencein the three groups.The mean migrationindex in response to the whole cell is indi-cated in Table V.DiscussionThe role of undernutrition with itsdepressive effect on CMI was assessedin APSGN. The patients and skin infec-tion controls were in the same nutritionalrange and most of the patients were nor-mal weight for age or had mild under-nutrition.Delayed cutaneous hypersensitivity toDNCB was normal in our patients. Theproportions of early and total T-rosetteswere normal in patients and skin infec-tion control in our study. Williams et al.in 1977(10) in their study on surface mar-ker  in APSGN reported similar findings.Subsequently, in 1981(11) they reportedthat T-cells were decreased during theacute stage when compared to skin infec-tio  controls, but was not different from619RAJAJEE ET AL. CELL MEDIATED IMMUNITY IN GLOMERULONEPHRITISnormal controls. This finding of lowT-cells during acute phase was thought toreflect hypo-reactivity to streptococcalantigens. Apart from finding normalT-cells numbers in patients and skin infec-tion controls, we also found good res-ponse to mitogen PHA and heterologousantigens PPD and BCG in all the 3groups of children in the lymphocytetransformation test. Bhat et al. (12) alsoreported normal lymphocyte transforma-tion response to mitogens in their studyon adult acute nephritis.Response to SK-SD and PPD waspresent in majority of the patients andcontrols in the indirect leucocyte migra-tion inhibition test. There was also nosignificant difference in the responseto the bacillarysuspensiono f  T1 2Group A ß-Hemolytic streptococcusbetween the patients, skin infection andnormal controls. Regarding response tostreptococcal membrane antigens, Bhatet al. (12,13) and Baldwin et al. (14) repor-ted that lymphocyte transformation wasdepressed in patients with acute nephritis.In both these studies, the patients wereadults with progressive disease. Theprognosis is different in children whencompared to adults and this could accountfor the different response in children (15).The normal controls in our study alsoresponded well to SK-SD and strepto-coccal antigens. This was probably be-cause most “normal” children belongingto the low socio-economic strata areexposed to scabies and impetigo infectedby various types of streptococci.Bes des,the bacillary suspension was used andhence there was multiple antigenic stimuli.In conclusion, cell mediated immunitywas normal in patients and skin infec-tion controls by all the parameters studiedand not significantly different from normalcontrols. Purified streptococcal antigen620could probably bring out a difference inresponse between controls and patients.REFERENCES1.2.3.4.5.6.7.8.9.10.11.12.Kaplan EL, Anthony BF, Chapman SS.Epidemic acute glomerulonephritis associatedwith type 49 streptococcalpyoderma. Am JMed 1970, 48: 9.Dillon HC, Reeves Mary S, Maxted WR.Acute glomerulonephritis following skininfection due to strep M Type 2. Lancet 1968,i: 533.White R. Diseases of the Kidney and UrinaryTract. In: Jeliffe and Morley (eds) Diseasesof Children in Subtropics and Tropics.Arnold Publications 1981, pp 433-434.Vinodhini Reddy, Bhaskaram C, Raghura-murlu N. Immunological responses in malnou-rished children. Indian Pediatr 1977, 14: 255.Chandra RK. Immunocompetence as afunctional index of nutritional status. BritishMed Bull 1981, 37: 89.Catalona WJ, Taylor PT, Robson AS,Cheetein PB. DNCB contact sensitisation.New Eng J Med 1972, 286: 399-402.Sarala Rajajee. Delayed cutaneous hyper-sensitivity to DNCB in iron deficiency ane-mia presented at the Indian Academy ofPediatrics Conference at Madras, Feb 1983.Wybran J, Shireen Chantler, Hugh Fruden-berg. Isolation of Normal T-cells in chroniclymphatic leukemia. Lancet 1973, i: 126.Jondal M, Helm G, Wigzell H. Surfacemarkers of T and B lymphocytes 1. A largepopulation of lymphocytes forming non-immune rosettes with sheep blood cells. JExp Med 1972, 136: 207.Williams RC (Jr), Zabriskie JB, Mabrose F,Hassaballa F, Godvin ZH. Lymphocytesurface markers in acute rheumatic feverand post streptococcal acute glomerulone-phritis. Clin and Exp Immunol 1977, 27:135-142.Williams RC (Jr), Van De Rijn I, Reid H,Poon T, King Zabriskie JB. Lymphocytecell sub-populations during acute post-strep-tococcal glomerulonephritis; cell surfaceantigens and binding of streptococcal mem-brane antigens and C-reactive protein. ClinExp Immunol 1981, 46: 397-405Bhatt JG, Gombos EA, Baldwin DS.Depressed cellular immune response toINDIAN PEDIATRICSstreptococcal antigens in post-streptococcalglomerulonephritis.Chin Immunopathol1977, 7: 230.13. Bhat JG, Gombos EA and Baldwin DS.Depressed CMI in response to streptococcalantigens in post-streptococcal glomerulone-phritis. Clin Immunol Immunopathol 1980,16: 48.14. Baldwin DS,Robert GS, Gloria RG,15.VOLUME 21 – AUGUST 1984Gluck MC, Feiner HD. Natural history ofpost streptococcal glomerulonephritis. In:Read SE, Zabriskie JB (eds} StreptococcalDiseases and the Immune Response. NewYork, Academic Press Inc. 1980, pp 563-579Rammelkamp CH. Acute Post-streptococcalglomerulonephritis. In: Read SE, ZabriskieJB (eds) Streptococcal Diseases and theImmune Response. New York, AcademicPress Inc 1980, pp 43-51.NOTES & NEWSDr. N.D. Datta Banik, Deputy Director General, Indian Councilof MedicalResearch has been elected as member of the American Academy of Pediatrics621

Cell mediated immunity in post-streptococcal glomerulonephritis

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