Cellular immune response to mitogens phytohemaggluthin (PHA) and poke

weed mitogen (PWM) was assessed in 13 patients with chyluria and 32 healthy

controls. The mean stimulation Index of the patient group was significantly lower

than the control group. The degree of depression was neither related to the duration

of excretion of chyle nor to the microfilaraemic status

Kumaraswami, V

Narayanan, P R

Raja, Alamelu

Thiruvengadam, K V

Tripathy, S P

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Indian J Med Res 77, April 1953, pp 443-446Cell-mediated immunity in chyluriaAlamelu Raja, V. Kumaraswamy, P.R. Narayanan,S.P. Tripathy and K.V. Thiruvengadam*Tuberculosis Research Centre and *Government General Hospital. MadrasRevised article received November 2, 1982Cellular immune response to mitogens phytohemaggluthin (PHA) and pokeweed mitogen (PWM) was assessed in 13 patients with chyluria and 32 healthycontrols. The mean stimulation Index of the patient group was significantly lowerthan the control group. The degree of depression was neither related to the dura-tion of excretion of chyle nor to the microfilaraemic status.Transformation of lymphocytes invitro induced by mitogens and antigens isa parameter commonly used to assesscellular immunity in patients with immuno-deficiency,auto-immunity and infec-tious diseases. Host immune responseto filarial infection has been under inten-sive study in the recent past. The variedclinical presentations found among indi-viduals in endemic regions have been relat-ed to different types of immunologicalresponses among infected individuals. Thecommon presentation of chronic ban-croftian filariasis in Madras includeslymphatic oedema of the limbs, hydro-cele and chyluria.Chyluria is the resultof obstruction and dilatation of the thoracicduct or its tributaries followed by therupture of distended lymphatics in theurinary tract1. To our knowledge, studiesin cellular immunity in patients withchyluria are meagre. We report in thiscommunication our observation that peri-pheral blood lymphocytes from chyluriapatients show marked reduction in res-ponse to phytohaemagglutinin (PHA) andpoke weed mitogen (PWM).Material and MethodsPatients for the study were chosen fromthose attending the Filariasis Clinic ofGovernment General Hospital, Madras.Thirteen patients with the complaint ofpassing milky urine were admitted to thestudy. The clinical history of the patientsis summarised in Table I. In addition,32 healthy subjects were included as con-trols.Lymphocytetransformation in vitro :Samples of peripheral blood were drawnfrom chyluria patients and volunteers, andlymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation.Cells were cultured in quadruplicate in0.2 ml of RPMI 1640 supplementedwith penicillin (100 IU/ml), streptomycin(100 µg/ml), glutamine (300 µg/ml) and10 per cent autologous plasma in 96-well443444 CMI in chyluriaTable 1. Transformation of lymphocytes by mitogens in 13 chyluria patients and 32 healthysubjects (control)Patient Sex/age Mf inno. bloodClinical status Duration Stimulation index*of chyleexcret ion PHA PWM(months)1 M/19 Episodes of fever, rigor andlymphadenitis2 M/22 -do-3 M/47 + v e -do-4 M/50 -do-5 M/20 -do-6 F/19 Inguinal lymphadenitis7 M/20 Fever, rigor, lympphadenitis, hydrocele8 M/18 – v e Fever, rigor, lymphadenitis9 M/21 Filarial fever10 M/28 Previous history of hydrocele11 M/30 No evidence of filariasis12 F/21 – v e -do-13 F/23 -do-Control subjects (mean of 32)ND, Not done. *For definition, see Material and Methods4 06 93 76 23 36 2618 012 012 336 1.53 02 02 3.545NDNDND70ND00144015315(U-bottom) tissue culture plate (Dyna-tech Laboratories Inc., Alexandria, Va)at a concentration of 0.5 x 106 cells/ml.PHA and PWM were added at a finalconcentrationof 1 µg/ml and 1/200dilution, respectively. These concentrationswere used, on the basis of our earlierstudy on human volunteers (unpublisheddata). Cultures were incubated at 37°C inan atmosphere at 5 per cent CO2 for 96 h.The proliferative response was measuredby adding 1.0 µCi of 3H-thymidine (Sp.act. 13000 mCi /mol, Bhaba AtomicResearch Centre, Bombay) and incubatingfor 18 h at 37°C.Cells were harvested with Mash II(Microbiological Associates, USA) anddeposited on Whatman filter paper. Paperdiscs were then transferred to biovialscontaining 2.5 ml of scintillation fluid (4 gof PPO and 0.5 g of POPOP in 1 litretoluene) and counted for 50 sec in a liquidscientillation counter (LSS-20, ECIL,India). Stimulation index (SI) was calcu-ated as follows :cpm in stimulated culturescpm in control culturesResults and DiscussionThe mean stimulation index of 32healthy subjects was 45 and 15 for PHAand PWM respectively. Eleven out of 13chyluria patients had a stimulation indexof less than 10 for PHA. The remainingRaja et al 445two had moderate stimulation indices of26 and 33. Considering PWM, 7 out of 9patients tested had an SI of 7 or less and inthe remaining 2 the values were normal.In a variety of parasitic infections such re-duction in the cellular immune responseshas been observed2-4. In the present studythe degree of depression of mitogen res-ponse was neither related to the durationof excretion of chyle nor to microfilaraemicstatus of the patient. This reduced res-ponse was not merely due to a shift inthe peak of dose or time kinetic curves,because the reduced response was seen atall time periods of the culture and withdifferent doses of mitogen studied (un-published data). The reduction in the res-ponse may not be due to humoral block-ing factors of lymphocyte activation, asthe addition of plasma from chyluriapatients does not reduce the responseof lymphocytes from healthy volunteersto mitogens.Representative data areprovided in Table II, similar results havebeen observed with plasma from a num-ber of chyluria patients. The absolutelymphocyte counts in the peripheralblood of chyluria patients were considera-bly reduced. Similar observations werelso reported by Date et al 5. The reductionin the mitogen response as such may notbe solely due to this reduction in theab olute lymphocyte counts because duringin vitro lymphocyte culture for bothhealthy subjects and chyluria patients, thecell suspensions are diluted appropriatelyin order to have similar number of lym-phocytes in each well of the microtitreplate. It is however possible that selectiveloss of mitogen responsive lymphocytesfrom the peripheral blood might haveoccurred. A similar phenomenon has beenobserved in intestinal lymphangiectasia6.Our attempts to collect and characterisethe lymphocytes from urine have not beensuccessful, as only 2 out of 13 patientshad lymphocytes in their urine.Our in vitro results supplement theobservations of Matsumoto et al 7 ofmarked reduction in skin test reactions toPPD, PHA and DNCB in chyluriapati nts. The mechanism of this immuno-suppresion is under further study.Table II. Blastogenic response of peripheral blood lymphocytes of a normal subject in presence ofnormal plasma and patient’s plasmaSource of plasmaMean counts/50 sec±SDControl PHA PWMAutologousNormal human plasmaChyluria plasma552 ± 138 32900 ± 11741 10127 ± 22581 705 ± 242 12645 ± 4594 6479 ± 17422 550 ± 193 4327 ± 3241 1906 ± 5831 1052 ± 346 24482 ± 3354 10565 ± 31972 697 ± 546 26606 ± 4858 11090 ± 4295Lymphocytes were from peripheral blood from a normal subjects446 CMI in chyluriaAcknowledgmentThe authors are thankful to Dr EricA. Ottesen, Laboratory of Parasitic Diseas-es, NIAID, National Institutes of Health,Bethesda, USA, Prof. S. Subramaniam,Head of the Department of Microbiology,Post-graduate Institute of Basic MedicalSciences, Madras and Dr M.S. Amaresan,Head of the Department of Nephrology,Government General Hospital, Madras,for their interest and encouragement duringthe study.References1. Philip Manson-Rahr- Manson’s tropical dis-eases, 16th ed. (Bailliere, Tindall and Cassel,London) 1955 p 681.2. Ottesen, E.A., Weller, P.F. and Heck, L.Specific cellular immune responsiveness inhuman filariasis. Immunology 33 (1977) 413.3. Grove, D.I. and Forbes, I.J. Immuno-suppression in bancroftian filariasis. Tran  RSoc Trop Med Hyg 73 (1979) 23.4. Mehta, K., Sindhu, R.K., Subramanyam, D.and Rao, C.K. Suppression of mitogenic res-ponse to PHA and Con A in bancroftianfilariasis. Indian J Med Res 72 (1980) 38.5. Date A., Padankatti, T., Vaska, P.H. andShastry, J.C. Lymphocytopenia in chyluria.Trans R Soc Trop Med Hyg 74 (1980) 137.6. Strober, W., Wochner, R.D., Carbone P.P.and Waldmen, T.A. Intestinal lymphangi-ectasia. J Clin Invest 46 (1967) 1643.7. Matsumoto, K., Tamaki, K. and Yamamoto,M. Clinico-immunological studies of filarialchyluria. Trop Med 22 (1980) 69.Reprint requests :Dr S.P. Tripathy, Director, Tuberculosis Research CentreChetput, Madras 600031

Cell-mediated immunity in chyluria

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