Role of protein kinase C and calmodulin in histamine release in experimental filariasis

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1 Allergology nternational (1998) 47: 3-8 Original Artile Role of protein kinase C and almodulin in histamine release in experimental filariasis Muralidhara P, Nany Mallo, Nirmal Kumar Ganguly* and Ramesh Chander Mahajan Departments of Parasitology and *xperimental Mediine and Biotehnology, Postgraduate nstitute of Medial duation and Researh, Chandigarh, ndia ABSTRACT The allergeni ativity of 6 kda antigen of Brugia malayi adult worm, due to its ability to release histamine from mast ells, has been previously reported by us. To determine the roles of protein kinase C (PKC) and almodulin in this proess, histamine release from passively sensitized lung and peritoneal mast ells was investigated in the presene of W-7 and trifluoperazine, used as almodulin inhibitors, and H-7, used as a PKC inhibitor. The results show that H-7 is able to inhibit histamine release from both lung and peritoneal mast ells, thus suggesting a vital role of PKC in histamine release. However, lung and peritoneal mast ells exhibited a differential response to the almodulin inhibitors. The almodulin inhibitors prevented histamine release from peritoneal mast ells, but not from lung mast ells, thus suggesting that almodulin is not essential for histamine release by lung mast ells. Key words: Brugia malayi, almodulin, histamine, lung mast ells, Mastomys natalensis, peritoneal mast ells, protein kinase C. NTRODUCTON Parasite-speifi g and the release of histamine from sensitized mast ells by parasite antigens is thought to be responsible for the linial manifestations of lymphati filariasis, suh as lymphadenitis, lymphangitis and Correspondene: Dr Nany Mallo, Additional Professor, Department of Parasitology, PGMR, Chandigarh-1612, ndia. mail: <medinst@pgi.hd.in> Reeived 1 May Aepted for publiation 27 September elephontiosis.' The interation of parasite antigen with speifi g on the ell membrane is responsible for Ca 2 + influx, whih transdues extraellular signals into intraellular events by binding with speifi proteins, suh as almodulin (CaM).2 This also stimulates the enzyme phospholipase C (PLC) to hydrolyse phosphatidyl 1 inositol-4,s, diphosphate to form inositol triphosphate (P3) and diaylglyerol (DAG). These produts of phosphoinositide hydrolysis are important seond messengers in ell otivotion.:' nositol triphosphate is responsible for the mobilization of intraellular Ca 2 +. This inrease in intraellular Ca 2 + is required for the ativation of Ca 2 +CaM-dependent protein kinases. Diaylglyerol also ats as a seond messenger by ativating protein kinase C (PKC). Protein phosphorylation atalysed these kinases is a prerequisite for the release of granule ontents from mast ells." However, there are few studies into the mehanisms involved in histamine release from mast ells during allergi manifestations of filariasis. n an earlier study, we demonstrated that the 6 kda antigen fration from adult Brugia malayi is an effiient allergen, as indiated by its apaity to release histamine from speifially sensitized mast ells, and it therefore may have an important role in the pathology of the diseose." The present study was designed to investigate the roles of CaM and PKC in the release of histamine from sensitized mast ells. This was done indiretly by the use of trifluoperazine (TFP) and (N-6 amino hexyl- 1)-S-hloro- 1 naphthalene sulfonamide (W-7) as almodulin inhibitors and 1-S-isoquinolinesulfonyl 1-2 methylpiperazine dihydrohloride (H-7) as a PKC inhibitor. While TFP and W-7 inhibit Ca 2 +CaM-dependent protein phosphorvlotion.? H-7 inhibits ell ativity by binding to phospholipid-dependent PKC (PLD-PKC).7 by

2 4 P MURAL/OHARA T AL. Both lung and peritoneal mast ells (LMC and PMC, respetively) were obtained and were passively sensitized with immune serum olleted at various time intervals after infetion with B. ma/ayi L 3 larvae to highlight the possible differenes in their response. Furthermore, investigations into the mehanisms involved in the release of histamine by the 6 kda antigen fration may have important impliations in the study of the pathology of filariasis. MTHODS Animals and parasites Male Mastomys nata/ensis (GRA strain), 3-4 weeks of age, bred by onventional methods were used in the present study. Animals were infeted subutaneously with 1 L3 larvae of B. ma/ayi obtained from infeted Aedes aegypti mosquitos. A mirofilarial ount was performed on 2 ml blood olleted from the tail vein periodially from 9 days postinfetion onwrds." Blood olleted at various times postinfetion from the retro-orbital plexus was used for serum separation and serum was stored at -7 C till use. Preparation of rude worm extrat antigen Crude worm extrat antigen was prepared essentially as previously desribed.? Briefly, B. ma/ayi adult worms olleted from Mastomys were given extensive washing with phosphate-buffered saline (PBS; ph 7.2). These worms were lyophilized and suspended in extration buffer ( rnrnol/l HPS, 1 rnrnol/l glyine, ph 7.2), whih ontained phenyl methyl sulphonyl fluoride (2 rnrnol/l., Sigma Chemial Co., St Louis, MO, USA), leupeptin (.2 mmol/l) and DTA (1 rnrnol/] as protease inhibitors. Parasites were homogenized in a hand-held glass tissue homogenizer. The homogenate was inubated overnight at 4 C with onstant agitation and was then entrifuged at 1 9 for 1 h. The supernatant was used as the antigen after protein estimation. Charaterization and purifiation of the 6 kda antigen The rude worm antigen was separated by sodium dodeyl sulfate-polyarylamide gel eletrophoresis (SDS PAG) in a 4% staking gel/1% running gel in the presene of -meraptoethanol.lo The gel ontaining the protein orresponding to the 6 kda antigen, with standard moleular weight markers, was ut and maerated. This was suspended in PBS (ph 7.2) and dialysed against PBS overnight to remove SDS. This dialysed material was entrifuged at 1 9 for 3 min. The gel-free supernatant was olleted and used for the histamine release assay. solation of mast ells An enzyme-digested lung tissue ell suspension from M. nata/ensis was prepared aording to the method desribed by Fox et /. 11 with slight modifiations. Lung tissue was washed twie with PBS (ph 7.2) and was ut into 2- mm piees. These piees were washed twie with ml Ca 2 +_M g2+ -free Hank's balaned salt saline (CMF-HBSS) ontaining 2 mrnol/l HPS and 1.3 X 1-4 mol/l DTA (ph 7.2) to remove epithelial ells. This was followed by digestion with pronase (2 mg/ml) to obtain free ells. The residual fragments were mehanially disrupted by repeated syringing. Supernatants from enzymati digestion were pooled with ells obtained from mehanial disruption. The resulting suspension was rapidly filtered through a 1 ml syringe ontaining loosely paked nylon woo/. Cells were washed with CMF-HBSS. Mast ells thus obtained were further purified by flotation through a disontinuous Peroll gradient and were entrifuged at 9 for 1 min. The ells found between the and 7% Peroll interphase were washed twie with CMF-HBSS. Mast ells obtained from peritoneal washings with CMF-HBSS were also washed and purified by Peroll gradient. The viability of isolated mast ells was heked with.1 % Trypan blue and purity was assessed by staining with.% Toluidine blue in. mol/l HC/. The viability and purity of the ells were> 9%. Histamine release assay Both LMC and PMC in HBSS (1-2 X 1 elis/ml) were pre-inubated at 3JOC for 1 min. Diluted immune serum in HBSS (2 ml) was added and inubated at 37 C for 3 min with gentle shaking to keep the ells in suspension. Mast ells reovered by entrifugation at 3 9 for 8 min were washed twie in HBSS. These ells were resuspended in HBSS at a onentration of 1 X 1 6 ells/ml and were used for the histamine release ossov.l? These presensitized ells were hallenged with 6 kda adult worm antigen (1 lg/ml) at 3JOC for 3 min. Subsequently, 2 ml ie-old CMF-HBSS was added and the ell suspension was entrifuged at 9 for 1 min.

3 HSTAMN RLAS N FLARASS The supernatant was assayed for histamine release. To assess total histamine, ells were lysed with.3 rnol/l perhlori aid, entrifuged and the supernatant assayed for histornine.':' Released histamine was expressed as a perentage and alulated as follows: % Released histamine = Histamine in supernatant X 1 Histamine in supernatant + histamine in lysate n vitro exposure of mast ells to blokers To study the role of PKC and CaM in the release of histamine, mast ells were pre-inubated at 37 C for 3 min with various drugs at different onentrations before being hallenged with the rude worm antigen. Different sets of experiments were performed for eah drug. A ell suspension without drug was taken as the ontrol. Preparation of drugs All drugs were purhased from Sigma Chemial Co. (St Louis, MO, USA). Trifluoperazine was dissolved diretly in HBSS and the ph was adjusted to 6.8. A stok solution of W-7 was prepared in HBSS at ph 2.3 with stirring in the dark at room temperature. Finally, the ph was adjusted to 6.8. A stok solution of H-7 was prepared in dimethyl sulfoxide (DMSO). Subsequent dilutions of H-7 were made in HBSS before use. None of the onentrations of solvents used interfered with the release and assay of histamine. Statistial analysis The results shown are representative data of six independent experiments. Various parameters were ompared by Student's unpaired t-test. RSULTS Serum samples olleted at various times postinfetion were used for the sensitization of mast ells before their ativation by 6 kda parasite antigen. t was found that serum olleted at months postinfetion indued optimum sensitization of both LMC and PMC to release maximum histamine (Figs 1,2). Hene, this serum was used for subsequent investigations to determine the role of various inhibitors in histamine release. xperiments investigating the inhibitory effet of H-7 on sensitized mast ells after ativation with 6 kda filarial antigen indiated that there was a signifiant derease in histamine release in both LMC and PMC in the presene Ql 3 Ql QJ 3 Ql 2 * 1 ** * Months postinfetion Fig. 1 Histamine release from sensitized lung mast ells after exposure to 6 kda Brugia malayi adult worm antigen. Mast ells were passively sensitized with serum olleted from mastomys at various times postinfetion and were hallenged with 6 kda antigen. (e), infeted; (), uninfeted ontrol. Data are the mean ± SD (n = 6). *No signifiant differene at the.1 level (P >.); ** signifiant differene at the.1 level (P <.1) Months postinfetion Fig. 2 Histamine release from sensitized peritoneal mast ells after exposure to 6 kda antigen of Brugia malayi at various times postinfetion. (e), infeted; (), uninfeted ontrol. Data are the mean ± SD (n = 6). "No signifiant differene at the.1 level (P >.); ** signifiant differene at the.1 level (P <.1).

4 6 P MURAL/OHARA T AL. of H-7 at onentrations as low as 1 11m (P <.1; Figs 3,4). The histamine release assay was performed in the presene of W-7 and TFP as CaM inhibitors at an optimum onentration of 2 urnol/l, t was found that W-7 signifiantly redued histamine released by 6 kda antigen (P <.1) from PMC at a minimum onentration of 2l1m W-7 (Fig. ). n ontrast, it was observed that W-7 did not inhibit histamine release from LMC at the same onentration, indiating differenes in the response by the two ell types in the presene of W-7 (Fig. 6). 6 ' Fig. 3 nhibition of histamine release in the presene of H-7. Mast ells were passively sensitized with immune serum and were inubated with various onentrations of H-7 at 3rC for 3 min. Subsequently, ells were hallenged with 6 koa antigen. A ell suspension without H-7 was taken as the ontrol. (e), infeted; (), ontrol. Results are the mean ± SO (n = 6). *No signifiant differene at the. level (P >.); **a signifiant differene at the.1 level (P <.1) [H-7] [pmol /L] o OL-_--_--..L---' --l [W-7] [prnof/l] ** 3 3 Fig. nhibition of histamine release in the presene of W-7. Mast ells were passively sensitized with immune serum and were inubated with various onentrations of W-7 at 3r. Subsequently, ells were hallenged with 6 koa antigen. A ell suspension without W-7 was taken as the ontrol. (e), infeted; (), ontrol. Results are the mean ± SO. *P >.; **P <.1. 8 e 6 2 * * 6 2 ** ** 2 4 Months postinfetion Months postinfetion 6 Fig. 4 Comparison of histamine release from lung mast ells (m) and peritoneal mast ells (i) after exposure to 6 koa antigen of Brugia ma/ayi in the presene of H-7. Values are the mean ± SO. *P >.. Fig. 6 Comparison of histamine release from lung mast ells (a) and peritoneal mast ells (ll) after exposure to 6 koa antigen of Brugia ma/ayi in the presene of W-7. Values are the mean ± SO; *P >., **P <.1.

5 HSTAMN RLAS N FLARASS a <l <l.g 2 2 Fig. 7 Comparison of histamine release from lung mast ells (W) and peritoneal mast ells (ll) after exposure to 6 kda antigen of Brugia ma/ayi in the presene of trifluoperazine. Values are the mean ± SD; *P >., **P <.1. xperiments using TFP showed no inhibition of histamine release from LMC at the fifth month postinfetion in both test and ontrol. However, there was a signifiant inhibition of histamine release in the presene of TFP (P <.1) when experiments were onduted with PMC. Thus, differenes in histamine release between the two ell types in the presene of TFP were statistially signifiant (Fig. 7). DSCUSSON 4 Months postinfetion Various pharmaologial agents have been used to study the roles of CaM and PKC in ell ativation, inluding TFP and W-7, whih inhibit Ca 2 +CaM-dependent protein phsphorvlotion.e":" and H-7, whih inhibits ell ativity by binding to PLD-PKC.7 The speifi inhibitory ativity of H-7 of PLD-PKC has also been used by others to demonstrate the role of PLD-PKC in the phosphorylation of proteins.pl" The ativation of mast ells by parasite antigen in the presene of these drugs helps to investigate the ativity of PLD-PKC or CaM and, hene, their roles in mast ell ativation. Although various methods are available for the diret measurement of PKC and CaM after their extration from the ell soure using speifi substrates, these methods are expensive and tedious to perform. Beause in the present study histamine release was taken as a marker of mast ell ativation, speifi drugs were used to inhibit histamine release and to establish the roles of PLD-PKC and CaM in mast ell ativation by parasite antigens. ** 6 The histamine release assay was performed using two prototype mast ell populations. Lung mast ells, belonging to the ategory of muosal mast ell (MMC), are also termed 'atypial mast ells' and PMC, whih represent onnetive tissue mast ells (CTMC), are termed 'typial mast ells'. These groups of mast ells differ in various properties, suh as their origin, staining properties, proteoglyan ontents and T ell dependene. 19 Thus, a parallel study was onduted, using both types of mast ells, in order to distinguish any possible differenes in the relative role of these two ell populations in the pathogenesis of filariasis. However, the results of an in vitro assay system in the present study annot be extrapolated to in vivo onditions, as the sensitization of these mast ells was performed artifiially under in vitro onditions. The present study was performed with the 6 kda parasite antigen at various times postinfetion. t was observed that the 6 kda antigen indued optimum histamine release at months postinfetion, whih was subsequently used for further studies. t was observed that H-7, a PLD-PKC inhibitor effetively bloked histamine release from both LMC and PMC ativated with 6 kda. This suggested the importane of PLD-PKC in histamine release. Similarly, Clark et 1.,2 while working with T ells, demonstrated that H-7 inhibited antigen or interleukin-2 indued proliferation of T ells. However, the possible involvement of other PKC, suh as yli nuleotide kinases, in the release of histamine from mast ells during filarial infetion annot be ruled out, as H-7 also inhibits the ativity ofthese PKC in addition to PLD-PKC. To determine the role of CaM in mast ell ativation by 6 kda antigen, ells were pre-inubated with TFPor W-7 before hallenge. There was a variation in the results obtained for both LMC and PMC in the presene of these inhibitors. While inubation with CaM antagonists had no effet on LMC, histamine release was bloked in PMC after ativation with 6 kda antigen. This suggests that CaM is a prerequisite for PMC ativation in filarial infetion, although the same is not true for LMC. This further suggests that the relative role of CaM or PLD-PKC is dependent on the type of ell as well as the nature of the ell ativator. Thus, from the present study, it is apparent that CaM-dependent PKC and PLD-PKC at through independent pathways. Similar results were obtained when Howraft et a/. 21 studied the role of PLD-PKC and CaM in the lyti ativity of primary ytotoxi T lymphoytes (CTL) by using speifi inhibitors, suh as H-7 and W-7. They observed that primary killer ativity of CTL required the ativity of both PLD-PKC- and CaM-dependent PKC,

6 8 P MURAL/OHARA T AL. whereas highly lyti ultured T ells required only CaM for ell ativation. Similarly, H-7 failed to inhibit the respiratory burst ativity and seretory response of stimulated neutrophils, whereas W-7 ompletely bloked neutrophil tivtion.i? Therefore, it is possible that a synergisti ation between DAG, whih ativates through PLD-PKC,3 and higher ytosoli Ca 2 +, whih ats through CaM, may be operating in mast ells. 23 Thus, it an be onluded from the present study that CaM-dependent pathways are not essential for LMC ativation, although they are absolutely essential for PMC ativation by the 6 kda antigen of B. ma/ayi. However, PLD-PKC is an essential requirement for both populations of mast ells as has already been demonstrated in rat mast ells." Further studies are needed to orrelate these in vitro findings to the in vivo situation in order to understand the role of these biohemial pathways in the pathogenesis of filariasis. RFRNCS Ottosen A, Neva FA, Paranjape RS, Tripathy Sp' Thiruvengadam KV, Beaven MA. Speifi allergi sensitization to filarial antigens in tropial eosinophilia syndrome. Lanet 1979; 1: Douglas WW, Nemeth F. On the alium reeptor exoytosis: nhibitory effets of almodulin-interating drugs on rat mast ells. J. Physiol. 1982; 323: Berridge MJ, rvine RF. nositol phosphate, a novel seond messenger in ellular signal transdution. Nature 1984; 313: Marone G. The role of mast ells and basophil ativation in human allergi reations. ur. Respir. J. 1989; 2 (Suppl. 6): S446-. Muralidhara P, Malia N, Ganguly NK, Mahajan RC. Response of mast ells against filarial antigens from experimentally infeted Mastomys natalensis with Brugia malayi. nt. Arh.AllergyAppl. mmunol. 1992; 98: Tanaka T, Ohmura T, Tamakaoo T, Hidaka H. Two types of alium-dependent protein phosphorylation modulated by almodulin antagonists naphthaline sulfonamide derivatives. Mol. Pharmaol. 1982; 22: Hidaka H, nagaki M, Kawamoto S, Sasaki Y. soquinolinesulfonamides, novel protein inhibitors of yli nuleotide-dependent kinase and protein kinase. C1in. Biohem. 1984; 23: Murthy Kp, Tyagi K, Roy Chowdhury TK, Sen AB. Suseptibility of Mastomys natalensis (GRA strain) to a subperiodi strain of Brugia malayi. ndian J. Med. Res. 1983; 77: Freedman DO, Nutman TB, Ottesen A. Protetiveimmunity in Banroftian filariasis. J. C1in. nvest. 1989; 83: Laemmli UK. Cleavage of strutural proteins during the assembly of head of bateriophage T 4. Nature 197; 227: Fox CC, Dvorak AM, Peters Sp' Kagey-Sobotka A, Lihtenstein LM. solation and haraterization of human intestinal muosal mast ells. J. mmunol. 198; 13: Nemeth F, Douglas WW. Differential inhibitory effets of the arahidoni aid analogue TYA1 on rat mast ell exoytosis evoked by seretagogues utilizing ellular or extraellular alium. ur. J. Pharmaol. 198; 67: Shore P, Burkhalter A, Cohn UH. Method for f1uorometri assay for histamine in tissues. J. Pharmol. xp. Ther. 199; 127: Hegemann L, Rooijer LM, Traber J, Shmidt BH. Polymyxine B is a seletive and potent antagonist of almodulin. ur. J. Pharmaol. 1991; 27: to A, Sato T, Ojima Y, Chen L, Nagase G, Mori Y. Calmodulin differentially modulates the interleukin- 1 indued biosynthesis of tissue inhibitor of metalloproteins and matrix metalloproteinases in human uterine ervial fibroblasts. J. Bioi. Chem. 1991; 266: Heller R, Bussolino F, Ghigo et 1. PKC and yli AMP modulate thrombin indued platelet-ativating fator synthesis in human endothelial ells. Biohem. Biophys. Ata 1991; 193: Huira M, Ozawa M, Ohtsuka T et 1. Stimulation of superoxide anion prodution in guinea pig polymorphonulear leukoytes by hypotoni onditions in ombination with protein kinase C ativators. Arh. Biohem. Biophys. 1991; 291: Kantengwa S, Polio BS. Queretin and karmpferol have potent protein kinase C inhibition ativity whereas staurosporine and H-7 are more speifi protein kinase C inhibitors. Biohem. Biophys. Res. Commun. 1991; 18: Clark RB, Love H, Sgroi, Lingenheld G, Shaffi R. Protein kinase C inhibitor, H-7, inhibits antigen and L-2 indued proliferation of murine T-ell lines. Biohem. Biophys. Res. Commun. 1987; 14: Howroft TK, Tatum SM, Cosgrove JM, Lindquist RR. Protein kinase C (PKC) inhibitors inhibit primary (1-) but not seondary (1-1) or loned ytotoxi T lymphoytes. Biohem. Biophys. Res. Commun. 1988; 14: Wright CD, Hoffman HMO. The protein kinase C inhibitors H-7 and HG fail to inhibit human neutrophil ativation. Biohem. Biophys. Res. Commun. 1986; 13: Katakami Y, Kaibuhi K, Sawamura M, Takai Y, Nishizuka Y. Synergisti ation of protein kinase C and alium for histamine release from rat peritoneal mast ells. Biohem. Biophys. Res. Commun. 1984; 121: 73-8.