Pro-inflammatory effect in mice of CvL, a lectin from the marine sponge Cliona varians

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Pro-inflammatory effect in mice of CvL, a lectin from the

marine sponge Cliona varians

Alexandre F.S. Queiroz

a

, Raniere M. Moura

b

, Jannison K.C. Ribeiro

c

, Ibson L. Lyra

c

,

Dayse C.S. Cunha

c

, Elizeu A. Santos

c

, Maurício. P. de-Sales

c,

⁎

a_{Departamento de Biofísica e Farmacologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil} b_{Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, CE, Brazil}

c_{Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Campus Universitário, 59072-970, Natal, RN, Brazil}

Received 21 August 2007; received in revised form 18 September 2007; accepted 18 September 2007 Available online 26 September 2007

Abstract

CvL, a lectin from the marine sponge Cliona varians agglutinated type A papainized erythrocytes and was strongly inhibited byD-galactose and sucrose. Models of leukocyte migration in vivo were used to study the inflammatory activity of CvL through of mouse paw oedema and peritonitis. Effect of CvL on peritoneal macrophage activation was analysed. Effects of corticoids and NSAIDS drugs were also evaluated on peritonitis stimulated by CvL. Results showed that mouse hind-paw oedema induced by subplantar injections of CvL was dose dependent until 50 μg/cavity. This CvL dose when administered into mouse peritoneal cavities induced maxima cell migration (9283 cells/μL) at 24 h after injection. This effect was preferentially inhibited by incubation of CvL with the carbohydratesD-galactose followed by sucrose. Pre-treatment of mice with 3% thioglycolate increases the peritoneal macrophage population 2.3 times, and enhanced the neutrophil migration after 24 h CvL injection (75.8%, pb0.001) and no significant effect was observed in the presence of fMLP. Finally, pre-treatment of mice with dexamethasone (cytokine antagonist) decreased (65.6%, pb0.001), diclofenac (non-selective NSAID) decreased (34.5%, pb0.001) and Celecoxib (selective NSAID) had no effect on leukocyte migration after submission at peritonitis stimulated by CvL, respectively. Summarizing, data suggest that CvL shows pro-inflammatory activity, inducing neutrophil migration probably by pathway on resident macrophage activation and on chemotaxis mediated by cytokines.

Keywords: Marine sponge; Cliona varians; Leukocyte migration; Paw oedema; Peritonitis

1. Introduction

Lectins are ubiquitous (glyco)proteins of plant or animal origin possessing an ability to specifically bind carbohydrate moieties including cells (Dodd et al., 1968). Some lectins from plant origin, can mimic endogenous mammalian lectins and trigger in vivo and in vitro migration of specific leukocytes migration in various inflammation models (Alencar et al., 2005a; Alencar et al., 2005b; Coelho et al., 2006; Napimoga et al., 2007).

The inflammatory process is a phenomenon that involves vascular and cellular events and can be defined as a generalized, nonspecific but beneficial response of tissues to injury due to

bacteria, virus, and/or parasites, chemical irritants, ultraviolet light irradiation, and nondigestive particles (O'Byrne et al., 2000; O'Byrne and Dalgleish, 2001; Dalgleish and O'Byrne, 2002), which can initiate this process that leads to chronic and acute diseases in mammalians. This complex process involves a variety of leukocyte type cells (macrophage, mononuclear cells and neutrophil) and probably dozens of inflammatory mole-cules, such as nitric oxide (NO), pro-inflammatory cytokines (TNF-a, IL-1, IL-12, IFN-γ), and chemokines (Key et al., 1982; Adams and Hamilton, 1984). Adhesion to the endothelium is a prerequisite for the movement of leukocytes from blood into affected tissues (Colditz 1985), that is mediated by membrane lectins in association with carbohydrate moieties present in specific leukocytes, providing the adhesion to vascular endo-thelial cells and the emigration of leukocytes to tissues, that is a feature of the inflammatory process (Tedder et al., 1995).

Comparative Biochemistry and Physiology, Part C 147 (2008) 216–221

www.elsevier.com/locate/cbpc

⁎ Corresponding author. Tel.: +55 84 32153415; fax: +55 84 32119208. E-mail address:msales@cb.ufrn.br(M.P. de-Sales).

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In marine animals, only a few phyla have been screened for the presence and distribution of lectins. In particular, the number of lectins that have isolated from invertebrate organisms is quite small as compared with the great variety of lectins isolated from plant origin and have been limited to those partially character-ized from molluscs and crustaceans (Miarons and Fresno, 2000). Since the discovery of hemagglutinins in sponges byDodd et al. (1968), there have been some reports on the purification and partial characterization of lectins from the oldest multicellular animals (Bretting and Kabat, 1976; Diehlseifert et al., 1985; Kamiya et al., 1986; Kamiya et al., 1990).

The biotechnological potential of Cliona varians sponge,

based in proteins, was first showed by us whenMoura et al.

(2006)demonstrated that a lectin purified from C. varians (CvL) displayed a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus and agglutinated Leishmania chagasi promastigotes. These findings were indica-tive of the physiological defense roles of CvL and its possible use in the antibiosis of bacteria and protozoa pathogenic.

In this study we purified a lectin from invertebrate of marine origin and investigated its effect on the neutrophil migration through in vivo inflammation models and its possible involvement in peritoneal macrophages and emigrant neutrophils activation. 2. Materials and methods

2.1. Materials

The Animal Care Unit of the Universidade Federal do Ceará, Fortaleza, CE, Brazil supplied Winstar mice, weighing 25–35 g. The animals had free access to food and water and were kept in standardized environmental conditions on a 12/12-h light/dark cycle. Before each test, the animals were fasted for at least 12 h.

Adult specimens of the marine sponge C. varians were collected in the littoral of Santa Rita beach, Extremoz located at Rio Grande do Norte state, Brazil. Specimens were collected and transported in ice to the laboratory and stored at 20 °C until use.

2.2. Purification of marine sponge C. varians lectin (CvL)

Methodology developed byMoura et al. (2006)was used to

purify CvL from C. varians marine sponge. The haemagglu-tinating activity was assayed in microtiter V plates (Nunc Brand products, Denmark) according to a twofold serial dilution

procedure (Debray et al., 1981). The erythrocytes used were

treated with papain according toBenevides et al.(1998). 2.3. CvL-induced rat paw oedema

Acute inflammation was tested on oedema induced by CvL in mice. Animals, allocated to treatment in groups of six, were injected into the plantar surface of the right hind paw of the mice (100μL containing 5, 10 and 50 μg of CvL, dissolved in 0.15 M

NaCl). While the contralateral paw was injected with 100μL

saline solution. Carrageenan (0.1 mL of a 1% 0.15 M NaCl) was injected into the plantar surface of the right hind paw of the mice

(control). The paws were amputated at the tarsocrural joint and

weighted on an analytical balance (Freire et al., 2003) and

difference between the left and the right paw oedema indicated the degree of inflammation after a period of 4 h. The average (mean ± S.E.M.) increase in paw oedema of each group was calculated.

2.4. Stimulation of leukocyte migration into peritoneal cavities by CvL

CvL was injected intraperitoneally (i.p.) at 50 μg/cavity in 0.1 mL of 0.15 M NaCl in six mice. Two control groups were made with six mice each: negative control group received the same volume of saline and positive control group received 100 ng of fMLP in substitution of the lectin solution. Four, 12, 24, 48, 72 and 96 h later, animals were sacrificed and peritoneal cells harvested by washing each peritoneal cavity with 3 mL of saline containing 5 UI/mL heparin. Total cell counts were performed as described elsewhere (Desouza and Ferreira, 1985). The results were reported as mean ± S.E.M. of the number of cells per mL of peritoneal wash. The average (mean ± S.E.M.) of each group was calculated.

2.5. Effect ofD-galactose,D-sucrose andD-fructose on the cell

migration induced by CvL

CvL (50μg) was dissolved in 100 μL of sterile saline solution containing either 0.2 MD-galactose or 0.2 MD-sucrose or 0.2 M

D-fructose and incubated by 30 min and injected into mice

peritoneal cavities. The effects were evaluated 24 h after injection and compared to the saline-treated control group and group treated with the carbohydrates. The average (mean ± S.E.M.) of each group was calculated.

2.6. Increase of the peritoneal macrophage population by treatment with thioglycolate

Thioglycolate (3%, w/v; 100μL i.p.) was injected into the peritoneal cavities and, after 4 days, peritoneal macrophages were collected, counted and compared to those from a group of non-treated animals (control) (Ribeiro et al., 1991). Saline (100μL /cavity) or CvL (50 μg/cavity in 100 μL of saline), was then injected into mice (control and Tg treated), and 24 h later, the neutrophil migration was evaluated. The average (mean ± S.E.M.) of each group was calculated.

2.7. Effect of pharmacological modulators on the neutrophil migration induced by CvL

The following drugs were used: (1) cytokine antagonist dexamethasone (0.5 mg/kg); (2) selective NSAID Celecoxib (2.8 mg/kg); (3) non-selective NSAID diclofenac (1 mg/kg). Control animals were injected i.p. with 1 mL of sterile saline. CvL (50μg/100 μL) was injected in the saline-treated animals and in animals previously treated with pharmacological modulators. Neutrophil migration was evaluated 4 h after injections and compared to the respective controls.

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2.8. Statistical analysis

All results were expressed as mean values ± S.E.M. for n experiments. Statistical evaluation was undertaken by analysis of variance (ANOVA) followed by Bonferroni's test for multiple comparisons. A p value of less than 0.001 was considered statistically significant.

3. Results

3.1. Induction of mice paw oedema by CvL

The injection of CvL (5, 10 and 50μg/paw) induced a dose-dependent oedema in paw mice when compared with the negative control group (0.15 M NaCl solution) (Fig. 1) and at

50μg/paw dose was observed higher oedema induction when

compared with positive control (1% Carrageenan solution). This dose was used in posterior assays.

3.2. Induced leukocyte migration into peritoneal cavities by CvL

The injection of CvL (50μg/cavity) gave an increase of 400%, of leukocyte migration, in acute phase (4 h post-injection), when compared with negative control (saline solution) and similar to fMLP at dose of 0.05μg/cavity. In chronic phase, the time–course curve had a maximal leukocyte migration at 24 h post-CvL injection, decreasing until 48 h, maintaining constant until 72 h and reducing leukocyte migration at 96 h after CvL injection, with level 10 times higher when compared to negative control (saline solution) (Fig. 2).

3.3. Effect of carbohydrate on CvL-induced leukocyte migration CvL, dissolved in 100μL of 0.2 MD-galactose and sucrose

andD-fructose solutions, was injected into the mouse peritoneal

cavity at the dose 50μg/cavity. After 24 h, results showed that

D-galactose nearly abolished the CvL-induced leukocyte

migration, followed by D-glucose and D-galactose migration.

On the other hand, sucrose partly (but not significantly) inhibited effect on CvL-induced leukocyte migration in peritoneal cavity (Fig. 3). When injected alone into the mice peritoneal cavity, these carbohydrates had no significant effect in the number of leukocyte compared with saline solution. 3.4. Thioglycolate treatment potentiates the neutrophil migration induced by CvL

The role of macrophages on the lectin-induced neutrophil migration was studied in vivo by altering in number the population of these cells. Intraperitoneal injection of 3% thioglycolate 72 h before in mice increased the macrophage population 2.3 times. The effect of administration of CvL (50μg/cavity), after treatment with thioglycolate, increased by 77% neutrophil migration in the peritoneal cavity (Fig. 4).

3.5. Cytokines antagonist (dexamethasone), but no selective NSAID (Celecoxib) inhibited neutrophil migration induced by CvL

Effect of the selective NSAID (Celecoxib), non-selective NSAID (diclofenac) and cytokine antagonist (dexamethasone) injected i.p 1 h before administration of the CvL on induction of neutrophil migration was tested. Selective NSAID (Celecoxib) had no effect, whereas non-selective NSAID (diclofenac) and dexamethasone inhibited significantly by 34.5% and 66%, respectively, the number of neutrophils that migrated to the peritoneal cavity of animals (Fig. 5).

4. Discussion

Bioactive proteins such as lectins have been isolated from various marine invertebrate, including the Porifera. The lectin, which was denominated CvL, exhibited papainized A erythrocyte

Fig. 1. Mouse paw oedema induced by CvL. Dose-dependent oedema with doses varying from 5 to 50μg/paw analysed 4 h after the injection of the stimuli (gray bars). Controls: 1% Carrageenan (black bar) and saline solution (white bar). The data are the mean ± S.E.M. of six mice. ⁎pb0.01 indicate that there were significant statistical differences compared to saline group (ANOVA– Bonferroni).

Fig. 2. Effect of CvL on cells migration into peritoneal cavity. Time course of leukocyte migration induced by CvL (50μg/cavity, ●) analysed at 4, 12, 24, 48, 72 and 96 h after the injections into peritoneal cavity. Controls: Saline solution (○) and fMLP (0.05 μg/cavity, ♦). The data were the mean±S.E.M. of six mice. The same letters indicate that there were no significant statistical differences, compared to saline group (pb0.01, ANOVA–Bonferroni).

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specificity and glycosylation and disulphide bridges in the protein indicating that CvL should be included in the C type lectin family,

such as Cinachyrella alloclada (Atta et al., 1989), Pellina

semitubulosa (Engel et al., 1992), Axinella polypoide (Buck et al., 1992) and Haliclona cratera (Pajic et al., 2002). The binding property of the CvL was found to be preferential forD-galactose.

A considerable number of marine invertebrate lectins, including

those isolated from sponges, were reported to react with D

-galactose (Bretting et al., 1981; Schröder et al., 1990) and this lectin type appears to have important roles in modulating immune responses in marine animals (Yousif et al., 1994; Mistry et al., 2001; Kurata and Hatai, 2002).

Various exogenous lectins, especially those from plant origin, demonstrated properties to active cells of the mammalian immune system, especially those with specificity to monosaccharidesD

-galactose andD-mannose (Bento et al., 1993; Benjamin et al.,

1997; Alencar et al., 2003). In the present work we have

inves-tigated effect of aD-galactose specific lectin from marine animal

origin on the in vivo leukocyte migration, an important cellular event of inflammation in mammalian. Following this propose, our results showed that CvL caused a significant dose-dependent

mouse paw oedema and that at dose of 50 μg/paw was

significantly higher than 1% Carrageenan used as positive control. Similar responses were reported for plant lectins from Artocarpus integrifolia (SantosdeOliveira et al., 1994), Ery-thrina velutina (Moraes et al., 1996) and Talisia esculenta (Freire et al., 2003). Inflammatory responses induced in mice by lectin from T. esculenta seeds (Freire et al., 2003), induced oedema effects in this type of inflammatory model. Also the effects of CvL on the in vivo leukocyte migration was evaluated and it was found that intraperitoneal injection of CvL (50μg/cavity) in mice caused significant acute leukocyte migration 4 h after injection as compared to saline group and was similar to fMLP group at dose

of 0.05μg/cavity. The maximal migration was observed at 24 h

Fig. 3. Inhibitory effect of carbohydrates on CvL-induced neutrophil migration into mice peritoneal cavity. Neutrophil migration was evaluated 24 h after CvL (50μg/ cavity) injection alone (black bar) or after incubation with 0.1 M of specific (D-galactose, sucrose and not specific carbohydrate (D-fructose) (gray bars). The white bars represent the neutrophil migration induced by the injection of the carbohydrate alone or saline solution. The data are the mean values ± S.E.M. of six rats. The same letters indicate that there were no significant statistical differences, compared with the results obtained in the group of animals which received CvL without incubation with the carbohydrate (pb0.01, ANOVA–Bonferroni).

Fig. 4. Analysis of the participation of macrophage on the neutrophil migration induced by CvL (50μg/cavity). (A) Peritoneal macrophage population in normal (saline) or pre-treated animals with 3% thioglycolate solution (Tg) after injection with 100μL saline. (B) Neutrophil migration induced by 100 μL saline (Sal), CvL (50 μg per cavity) and fMLP (0.05μg per cavity) in normal (saline) or 3% thioglycolate pre-treated animals (Tg). Results were means±S.E.M. (n=6). The same letters indicate that there were no significant statistical differences, compared to saline group ( pb0.01, ANOVA–Bonferroni).

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post-injection and this migration was significantly diminished

when monosaccharideD-galactose was added together with CvL

in the peritoneal cavity, indicating that its carbohydrate binding site can mediate this pro-inflammatory effect. The participation of macrophages on the induction of CvL cells migration in mice peritoneal cavities was evaluated. Thioglycolate treatment raised the macrophage population 2.3 times causing elevation of neutrophil migration induced by CvL in 77%, but it did not modify the responsiveness to fMLP, a known direct chemoat-tractant. This novel finding showed that also a marine invertebrate lectin can modulate the neutrophil recruitment by indirect mechanisms like other exogenous lectins from plant origin, specially those purified from E. velutina (Moraes et al., 1996), Glycine max (Benjamin et al., 1997), T. esculenta (Freire et al., 2003), Vatairea macrocarpa (Alencar et al., 2003), Lonchocar-pus sericeus (Alencar et al., 2005a), and Pisum arvense (Alencar et al., 2005b) seeds.

The early stages of the inflammatory process, macrophages, mast cells and lymphocytes participate in the control of neutrophil migration. This control is mediated via release of chemotactic factors such as leukotrienes (Rankin et al., 1990), components of

the complement system (Whaley and Ferguson, 1981), and

cytokines, mainly interleukin-1 (IL-1), IL-8, TNF-α (Staruch and Wood, 1985; Rankin et al., 1990) and macrophages-derived neutrophil chemotactic factor (MNCF) (Cunha and Ferreira, 1986). Since CvL-induced neutrophil migration seems to follow an indirect pathway, mediated by macrophages, the possible mediators involved in this event were investigated, using selective NSAID (Celecoxib), non-selective NSAID (diclofenac) and cytokine antagonist (dexamethasone). Among these inhibitors, cytokine antagonist (dexamethasone) reduced significantly the neutrophil migration to the peritoneal cavity after stimulation by CvL. The dexamethasone inhibitory effect could be explained by the blockage of the release of chemotactic factors, stimulated by inflammatory stimuli (Ribeiro et al., 1997), especially IL-8 (Ribeiro et al., 1991). It is therefore postulated that these findings favour the

hypothesis that CvL-induced neutrophil migration could possibly be mediated via release of cytokines by resident macrophages, since these cells represent an important source of cytokines. The results reported here demonstrated that the galactose/sacarose specific lectin from C. varians sponge possesses a pro-inflamma-tory activity, which induces neutrophil migration probably via the release of cytokines from macrophages. These findings indicate that lectins can be used, as tools to better understand the mechanisms involved in inflammatory responses or cellular event of inflammation.

These findings showed that the isolation and characterization of proteins from marine animals have turned into a fast growing field in the life sciences. Many marine bioactive molecules are attracting more and more attention due to their extensive physiological, biological and pharmacological use. The impor-tance of marine biotechnology has grown immensely because of the achievements of these natural marine products.

Acknowledgments

This work was supported by Brazilian Agencies: FINEP, CAPES and CNPq. The authors thank Dr Rosana Lucena de Sá Leitão, Dr Dulce H. Seabra de Souza Silva and Silene Telma Lima de Santana, pharmacists from Hemonorte, Natal, RN, Brazil, for the generous blood bag donation.

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