Immunomodulatory role of substance P in the wall lizard Hemidactylus flaviviridis: An in vitro study

Neuropeptides 45 (2011) 323–328

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Immunomodulatory role of substance P in the wall lizard Hemidactylus flaviviridis: An in vitro study Sunil Kumar, Umesh Rai ⇑ Department of Zoology, University of Delhi, Delhi 110 007, India

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Article history: Received 23 May 2011 Accepted 2 July 2011 Available online 23 July 2011 Keywords: Reptiles Phagocytes Substance P NK-1 receptor AC-cAMP-PKA Phagocytosis

a b s t r a c t Present in vitro investigation for the first time in ectotherms demonstrated the immunomodulatory role of substance P in the wall lizard Hemidactylus flaviviridis. Substance P inhibited the percentage phagocytosis and phagocytic index of lizard splenic phagocytes. Inhibitory effect of substance P was completely blocked by NK-1 receptor antagonist spantide I, indicating the NK-1 receptor mediated action. Further, NK-1 receptor-coupled downstream signaling cascade involved in controlling phagocytosis was explored using inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). Both the inhibitors, in a concentration-related manner decreased the suppressive effect of substance P on phagocytosis. In addition, substance P treatment caused an increase in intracellular cAMP level in splenic phagocytes. Taken together, it can be suggested that substance P via NK-1 receptor-coupled AC-cAMP-PKA pathway modulated the phagocytic activity of splenic phagocytes in wall lizards. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Substance P (SP), a member of neurokinin/tachykinin peptide family is an eleven amino acid long peptide (Chang et al., 1971). It is widely expressed in central and peripheral nervous system of vertebrates, and thus regulates number of physiological functions (Hökfelt et al., 1977; Severini et al., 2002). The SP immunoreactive nerve endings are demonstrated in the close proximity of immune cells in lymphoid organs of mammals (Bellinger et al., 1990; Felten and Felten, 1991; Lorton et al., 1991; Romano et al., 1994), indicating the involvement of this peptide in immunomodulation. Although direct role of SP in controlling non-specific (Bar-Shavit et al., 1980; Jeon et al., 1999; Ahmed et al., 2001; Rogers et al., 2006; Sun et al., 2008) and specific (Stanisz et al., 1986) immune responses is reported in mammals, these studies are totally lacking in non-mammalian vertebrates including reptiles. In the present in vitro investigation we tried to explore the immunoregulatory role of SP in a phylogenetically important group, reptile, the common ancestors to both birds and mammals. Due to easy availability and maintenance, the Indian wall lizard Hemidactylus flaviviridis that belongs to class reptilia was used in the present in vitro study. Members of tachykinin peptide family mediate their effect through tachykinin/neurokinin receptors which are classified into ⇑ Corresponding author. Tel.: +91 11 27667443, 27667212x106; fax: +91 11 27667985. E-mail address: [email protected] (U. Rai). 0143-4179/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2011.07.001

three types, neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 (NK-3) receptors (Patacchini and Maggi, 1995; Pennefather et al., 2004). While SP shows higher binding affinity for NK-1, the NK-2 and NK-3 receptors preferentially bind with other tachykinins, neurokinin A and neurokinin B, respectively (Liu and Burcher, 2005). Neurokinin receptors are demonstrated on different cell types of vertebrates by cloning (Yokota et al., 1989; Takahashi et al., 1992; Liu et al., 2004), radioligand binding assay (Too and Hanley, 1988; Weld et al., 1994; Liu and Burcher, 2001) and pharmacological (Akasu et al., 1996) techniques. These receptors are the G-protein-coupled receptors (GPCRs) (Nakanishi, 1991; Maggi, 1995; Pennefather et al., 2004) that on ligand binding commence various downstream signaling cascades (Nakajima et al., 1992). Nonetheless, studies pertaining to neurokinin receptor-coupled downstream signaling cascades are confined to mammals, and so far no report is available in non-mammalian vertebrates including reptiles. Thus, in the present in vitro study, an attempt was made to pharmacologically demonstrate the existence of functional neurokinin receptor on lizard splenic phagocytes. Also, neurokinin receptor-coupled downstream signaling cascade involved in mediating SP action was investigated. In ectothermic vertebrates, the adaptive immune responses take time to respond against invading pathogens (Neumann et al., 2001), under the circumstances, the innate immunity plays important role to combat the pathogenic attack. Phagocytes including macrophages and neutrophils are the effector cells of innate immune responses and form the first line of host defense against microbial infections. These cells limit the initial

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dissemination and growth of pathogens by phagocytosis and releasing cytotoxic substances. Considering phagocytosis as an important function of phagocytes in ectotherms, role of SP in controlling phagocytic activity of splenic phagocytes was investigated in the wall lizard H. flaviviridis. 2. Materials and methods 2.1. Animals Common Indian wall lizard H. flaviviridis of adult age (8–10 g body weight and 5–6 cm snout–vent length) were collected from suburbs of Delhi, India (Delhi: latitude: 28°120 –28°530 N, longitude: 76°500 –77°23) and housed in wooden cages with wire mesh at top and sides. Lizards were acclimated to the laboratory conditions (room temperature and 12L:12D with lights on at 07:00 h) for 1 week prior to experiments. They were provided with live house flies as feed and water ad libitum. Females were used in this study owing to their greater immune responses as compared to males (Mondal and Rai, 1999). The Institutional Animal Ethics Committee guidelines for the sacrifice and experiments on animals were followed. 2.2. Reagents and culture medium Cell culture medium RPMI 1640, inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89), MTT [3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], substance P (SP), neurokinin 1 receptor antagonist spantide I, and cAMP immunoassay kit were purchased from Sigma–Aldrich Co., (St. Louis, MO, USA). Molecular biology grade routine chemicals were purchased from Merck Specialties Pvt., Ltd., (Navi Mumbai, India), Sisco Research Laboratories Pvt., Ltd., (Mumbai, India), Qualigens Fine Chemicals (Mumbai, India), and Central Drug House (P) Ltd., (New Delhi, India). The cell culture medium was supplemented with 100 lg/ml streptomycin, 100 IU/ml penicillin, 40 lg/ml gentamicin, 5.94 mg/ml HEPES buffer {N-(2-hydroxyethyl) piperazine-N0 (2-ehtanesulphonic acid)} and 0.2% sodium bicarbonate. Prior to use, 2% heat-inactivated fetal calf serum (FCS) (Biological Industries, Kibbutz Beit, Haemek, Israel) was added to cell culture medium and referred as complete culture medium. The stock solutions (1 mg/ml) of SP and NK-1 receptor antagonist spantide I were made in acidic deionized water (0.05 M acetic acid) and phosphate buffered-saline (PBS, pH 7.2), respectively. Both the stock solutions were serially diluted in PBS, and the final required concentration(s) of SP and spantide I were prepared in complete culture medium prior to use. 2.3. Preparation of splenic phagocyte monolayer The method of Mondal and Rai (1999) was followed to prepare the splenic phagocyte monolayer. In brief, wall lizards were sacrificed by cervical dislocation, pinned, and dissected in aseptic conditions. The spleens were excised and pooled in chilled PBS. To prepare the single cell suspension of splenocytes, spleens were forced to pass through nylon mesh (pore size 90 lm) in ice-cold complete culture medium. After adjusting the cell number to 106 cells/ml, 200 ll of splenocyte suspension was flooded on each pre-washed slide. Following 90 min of incubation, non-adherent cells were washed off with PBS. The viability of adhered phagocytes was above 98% as assessed by trypan blue exclusion method. All the incubations were carried out at 25 °C (±1) in humidified chamber/incubator maintained with 5% CO2.

2.4. Preparation of yeast cell suspension The yeast cell suspension was prepared by warming the commercially available Baker’s yeast (Saccharomyces cerevisiae) (1.5 mg/ml PBS) for 20 min at 80 °C. Afterwards, heat-killed yeast cell suspension was washed and resuspended in the complete culture medium. 2.5. Phagocytic assay For phagocytic assay, splenic phagocyte monolayer on each slide was incubated with 400 ll yeast cell suspension. After 90 min, non-phagocytosed yeast cells were washed off with PBS. The monolayer was fixed in methanol, stained with Giemsa and mounted in DPX. The cells were observed under the microscope (Nikon Eclipse E400) at 400 magnification. Phagocytes engulfing one or more than one yeast cell were considered as positive phagocytes. These cells characteristically show the extended pseudopodia forming the phagocytic cup(s) around the yeast cell(s). Approximately, 200 phagocytes per slide were counted without any predetermined sequence or scheme. The experimenter was blind to the technical details of slides while counting. Percentage phagocytosis and phagocytic index were calculated using following formulae (Campbell et al., 2003): (a) percentage phagocytosis = number of positive phagocytes/100 phagocytes (b) phagocytic index = average number of yeast cells engulfed by each positive phagocytes  percentage phagocytosis. 2.6. In vitro experiments 2.6.1. Effect of substance P Splenic phagocytes were treated with different concentrations of SP ranging from 1011 M to 107 M for 30 min. Cells incubated in medium alone for the same time duration were considered as control. After incubation, phagocyte monolayer was washed with PBS and processed for phagocytic assay. As reports are lacking in reptiles, the literature available in other vertebrates was consulted to determine the range of SP concentrations to be used in this study (Jeon et al., 1999; Ahmed et al., 2001; Rogers et al., 2006; Sun et al., 2008). Pilot experiments were performed using lizard splenic phagocytes to decide the optimum in vitro incubation time for SP action. 2.6.2. Effect of neurokinin 1(NK-1) receptor antagonist In accordance to concentration-related effect of SP on phagocytic activity, splenic phagocytes were incubated with the most effective concentration of SP (108 M) and 10 times higher concentration of NK-1 receptor antagonist, spantide I (107 M), simultaneously, for 30 min. Spantide I is the competitive NK-1 receptor antagonist, therefore, splenic phagocytes were treated with spantide I and SP for the time period (30 min) which is optimum for the SP action on phagocytosis. For control, phagocytes were incubated in medium alone/108 M SP/107 M spantide I for the same duration. After incubation, cells were washed and processed for phagocytic assay. 2.6.3. Effect of inhibitors for adenylate cyclase and protein kinase A To delineate the receptor-coupled downstream signaling mechanism for SP action, splenic phagocytes were pre-incubated with different concentrations of SQ 22536 (2.5, 5.0, 7.5, and 10.0 nM)/ H-89 (25, 50, 75, and 100 nM) for 30 min. Afterward, cells were treated with 108 M SP and varying concentrations of SQ 22536/ H-89 for 30 min. Several control groups were made in which phagocytes were: (a) incubated in medium alone for 60 min (b) pre-incubated in medium alone for 30 min and then with 108 M SP for 30 min (c) incubated with 10 nM SQ 22536/100 nM H-89

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Fig. 1. Effect of SP on percentage phagocytosis and phagocytic activity of splenic phagocytes. Spleens of 10 lizards were pooled to prepare the phagocyte monolayer. Cells were treated with different concentrations of SP for 30 min. Phagocytes incubated in medium alone were considered as control. Each treatment was performed in triplicate. The experiment was repeated three times to verify the reproducibility of results. Data of three experiments were pooled, analyzed by one way analysis of variance (ANOVA), and represented as mean ± SEM. Error bars bearing different superscripts(ac/AC) differ significantly (Newman Keuls’s multiple range test, P < 0.01/0.05). Bar and line graphs represent values of percentage phagocytosis and phagocytic index, respectively.

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Fig. 3. Effect of NK-1 receptor antagonist spantide I on SP-induced-inhibition of phagocytic activity of splenic phagocytes. Spleens of 10 lizards were pooled, and processed for the preparation of phagocyte monolayer. Cells were treated with 107 M Spantide I and 108 M SP, simultaneously for 30 min. Each treatment was carried out in triplicate, and the experiment was repeated three times using different spleen samples (10 lizards/spleen sample). Data of three independent experiments were pooled, analyzed (ANOVA), and presented as mean ± SEM. Error bar bearing different superscripts(ab/AB) differ significantly (Newman Keuls’ multiple range test, P < 0.01).

in the supernatant using commercially available cAMP immunoassay kit (Sigma–Aldrich Co., St. Louis, MO, USA.). 2.8. MTT assay

a Absorbance at 570 nm

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for 60 min. After incubation, cells were washed with PBS and processed for phagocytic assay. The concentration range of the SQ 22536 and H-89 used were determined based on the literature available (Roy et al., 2008; Roy and Rai, 2009) and pilot experiments. 2.7. cAMP assay The manufacturer’s (Sigma–Aldrich, St. Louis, MO, USA) protocol was followed to estimate the intracellular cAMP content in splenic phagocytes treated with SP. In brief, phagocyte monolayer was prepared by adding 100 ll splenocyte suspension to each well of 96-well tissue culture plate. Following 90 min incubation, nonadhered cells were washed off, and adhered cells were incubated with phosphodiesterase inhibitor (IBMX, 104 M) for 30 min. Afterward, phagocytes were incubated in PBS (control)/108 M SP for 30 min. Cells were washed, lyzed with 0.1 M HCl and centrifuged at 600g for 10 min at room temperature. The cAMP was estimated

MTT assay is based on the ability of mitochondrial enzyme succinate dehydrogenase in live cells to reduce MTT into the membrane impermeable and insoluble purple crystals called formazan. The number of live cells is directly related to the amount of formazan produced. In this study, MTT assay (Mosmann, 1983) was performed to assess the effect of different concentrations of SP on viability of splenic phagocytes. Briefly, phagocyte monolayer was prepared by adding 100 ll splenocyte suspension to each well of 96-well culture plate. After washing the non-adhered cells, adhered phagocytes were incubated with varying concentrations of SP and medium alone (control) for 30 min. Phagocyte monolayer was washed and incubated in 100 ll of medium containing 0.5 mg/ml MTT for 2 h. Thereafter, cells were washed with PBS and permeabilized by adding 20 ll of 0.1% triton-X 100. The reduced product formazan was solubilized in 150 ll of DMSO. After incubation for 15 min at room temperature, the absorbance was measured at 570 nm using ELISA plate reader (MS 5608A, ECIL, India). 3. Statistical analysis Each treatment was performed in triplicate. All the experiments were repeated three times with different lizards. The data of three independent experiments were pooled and analyzed by one way analysis of variance (ANOVA) followed by Newman Keuls’ multiple range test and represented as mean ± SEM. In case of cAMP estimation, Student’s t-test was performed to compare the result of two groups. 4. Results 4.1. Effect of substance P SP at lower concentrations (1011 and 1010 M) did not affect the phagocytic activity of splenic phagocytes (control vs 1011/ 1010 M SP, non significant). However, a significant decrease in

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Fig. 4. Concentration-related effect of adenylate cyclase/protein kinase inhibitor, SQ 22536 (A)/H-89 (B), respectively, on SP-induced inhibition of phagocytic activity. Splenic phagocytes were treated with different concentrations of SQ 22536/H-89 for 30 min, and thereafter incubated with SP + varying concentrations of SQ 22536/H-89 for 30 min, simultaneously. Each treatment was carried out in triplicate and the experiment was repeated three times. Data of three independent experiments were pooled, analyzed by one way analysis of variance (ANOVA) and Newman Keuls’ multiple range test, (P < 0.01), and presented as mean ± SEM. Error bars bearing same superscript(ac/AC) do not differ significantly.

phagocytosis was observed at 109 M concentration of SP (109 M SP vs control, P < 0.01, Fig. 1). The inhibitory effect of SP was further pronounced at 108 M concentration (109 M vs 108 M, P < 0.01). With further increase of SP concentration to 107 M, no more escalation in SP-induced inhibition of phagocytosis was observed. SP at any of its concentrations did not affect the viability of splenic phagocytes as assessed by MTT assay (Fig. 2).

The neurokinin 1 (NK-1) receptor antagonist spantide I completely antagonized the inhibitory effect of SP on phagocytic activity when splenic phagocytes were incubated with SP and spantide I, simultaneously (108 M SP vs 108 M SP + 107 M spantide I, P < 0.01). The percentage phagocytosis and phagocytic index of cells treated with SP and its antagonist spantide I were seen comparable to that of incubated in medium alone (Fig. 3).

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SP

Fig. 5. Effect of SP on intracellular cAMP production in splenic phagocytes of wall lizards. Cells were treated with SP (108 M) for 30 min. The intracellular cAMP content was estimated using enzyme immunoassay. Data was analyzed by Student’s t-test (P < 0.01) and represented as (mean ± S.E.M).

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activity in a concentration-dependent manner (ANOVA, P < 0.001, Fig. 4). SQ 22536 at 5 nM (Fig. 4A) and H-89 at 50 nM (Fig. 4B), markedly reduced the inhibitory effect of SP on percentage phagocytosis and phagocytic index (108 M SP vs 108 M SP + 5 nM SQ 22536/50 nM H-89, P < 0.01). At higher concentrations (7.5/ 10 nM SQ 22536 or 75/100 nM H-89), both the inhibitors completely abrogated the inhibitory effect of SP on phagocytosis and the results were similar to that of control (medium alone) (Fig. 4A and B). 4.4. Intracellular cAMP As compared to control, SP treatment significantly (P < 0.01) increased the intracellular cAMP content in splenic phagocytes (Fig. 5).

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ported to regulate the expression of CD2 and IL-2 receptors on T-lymphocyte cell line Jurkat cells (Sudduth-Klinger et al., 1992). In the present in vitro investigation, suppressive effect of SP on phagocytosis was abrogated by AC inhibitor SQ 22536. Further, an increase in intracellular cAMP level was observed in SP-treated splenic phagocytes. It appears that in lizard splenic phagocytes, SP through NK-1 receptor activated the AC, and consequently intracellular cAMP level was increased. Since cAMP by modulating the PKA activity influences various cellular functions, implication of PKA in mediating SP effect was envisioned in the present study. Interestingly, PKA inhibitor H-89 decreased the inhibitory effect of SP on phagocytosis thereby confirming the involvement of PKA. Thus, it can be summarized that in the wall lizard H. flaviviridis, SP downregulated the phagocytic activity of splenic phagocytes via NK-1 receptor that are coupled with AC-cAMP-PKA signal transduction pathway.

5. Discussion Substance P, a member of tachykinin peptide family modulates innate as well as adaptive immune functions in vertebrates. Moreover, studies are confined to mammals (Bar-Shavit et al., 1980; Jeon et al., 1999; Ahmed et al., 2001; Sun et al., 2008) and till date no report is available in non-mammalian vertebrates including reptiles. Present in vitro study in reptile, for the first time in nonmammalian vertebrates, demonstrated the immunoregulatory role of SP. In the wall lizard H. flaviviridis, SP inhibited the phagocytosis of yeast cells by splenic phagocytes in a concentration-dependent manner. This is in contrast to human polymorphonuclear leukocytes (Bar-Shavit et al., 1980) and mouse peritoneal macrophages (Bar-Shavit et al., 1980) in which SP enhanced the phagocytosis of yeast cells (Saccharomyces cerevisiae). Similarly, Rogers et al. (2006) have observed an increase in phagocytosis of fluorescent Escherichia coli by bovine alveolar macrophages. Nonetheless, SP displayed a tendency to inhibit the phagocytic activity of murine macrophage cell line RAW 264.7 to phagocytose Leishmania major parasite, though the effect was insignificant (Ahmed et al., 2001). Our observation in wall lizard splenic phagocytes is in accordance to that observed by Ahmed et al. (2001). Taken together, it is likely that the effect of SP on phagocytosis, more or less depends on different kinds of target cells (yeast cells/Leishmania major parasite/ Escherichia coli) used. Nevertheless, more experiments across the vertebrate species using different target cells are required to develop a conceptual knowledge about the effect of SP on phagocytosis. Regarding receptor-mediated action, SP is shown to mediate its effect through NK-1 receptor (Patacchini and Maggi, 1995), though it also displays lower binding affinity to other NK receptors (Maggi, 1995). In the present in vitro investigation, using NK-1 receptor antagonist spantide I, we were able to pharmacologically demonstrate the existence of functional neurokinin receptor on lizard splenic phagocytes. Spantide I completely abrogated the inhibitory effect of SP on phagocytosis, indicating the involvement of NK-1 receptor in transducing SP effect on splenic phagocytes. This is in concurrence to studies in mammals, wherein, SP through NK-1 receptor modulated the immune functions (Jeon et al., 1999; Rogers et al., 2006; Douglas et al., 2008). Neurokinin receptors are the G-protein coupled receptors which are coupled with multiple downstream signaling cascades, such as, phospholipase C-protein kinase C (Mitsuhashi et al., 1992), adenylate cyclase-protein kinase A (Mitsuhashi et al., 1992; Nakajima et al., 1992), and ERK1/2 and p38 MAPK (Sun et al., 2008) pathways. With regard to immune responses, different downstream signaling cascades are involved in regulation of diverse immune functions. While ERK1/2-p38 MAPK pathway was shown to regulate cytokine production in murine peritoneal macrophages and macrophage cell line RAW 264.7 (Sun et al., 2008, 2009), Ca2+/calmodulin-dependent protein kinase pathway was re-

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