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Canscora decussata promotes adhesion of neutrophils to human umbilical vein endothelial cells
Journal of Ethnopharmacology 79 (2002) 229– 235 www.elsevier.com/locate/jethpharm
Canscora decussata promotes adhesion of neutrophils to human umbilical vein endothelial cells Babita Madan, Balaram Ghosh * Molecular Immunology and Immunogenetics Laboratory, Centre for Biochemical Technology, Uni6ersity of Delhi Campus (North), Mall Road, Delhi 110 007, India Accepted 1 November 2001
Abstract A regulated expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells is essentially required for immune surveillance. Cell adhesion molecules regulate the extravasation and migration of body’s effector cells to the site of immune activation. Failure of expression of cell adhesion molecules leads to various pathological diseases as it impairs the individuals’ ability to mount an inflammatory response. Our results demonstrate that an aqueous extract prepared from Canscora decussata (CdAqE), promotes the adhesion of peripheral neutrophils to human umbilical vein endothelial cells. The increased adhesion is as a result of induction of the expression of ICAM-1 and E-selectin on endothelial cells. CdAqE increases the expression of steady state transcript levels of these molecules as determined by RT-PCR. These results have implications for the usage of aqueous preparation of C. decussata for upregulation of cell adhesion molecule expression. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Canscora decussata; Endothelial cells; Neutrophils; Adhesion molecules; Immunomodulators
1. Introduction Lymphocytes continually patrol the body for foreign antigens and allow efficient surveillance of the tissues. In response to any injury or infection the endothelium gets activated and the leukocytes are recruited locally into the site by various chemotactic agents including lipopolysaccharide derived from bacterial cell walls, cytokines, and eicosanoids produced by local tissue monocytes and endothelial cells. A regulated expression of cell adhesion molecules such as ICAM-1 (intercellu-
Abbre6iations: ICAM-1, intercellular adhesion molecule-1; VCAM1, vascular cell adhesion molecule-1; TNF-a, tumor necrosis factor-a; LPS, lipopolysaccharide; IL-1, interleukin-1; Mac-1, CD11b/CD18; HUVECs, human umbilical vein endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; mAb, monoclonal antibody; PBS, phosphate buffer saline. * Corresponding author. Tel.: +91-11-766-7602; fax: + 91-11-7667471. E-mail address: [email protected] (B. Ghosh).
lar adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), and E-selectin on the endothelium controls the trafficking of the lymphocytes and their migration to the sites of injury or infection. These cell adhesion molecules are not expressed on unstimulated endothelial cells but their expression is induced on various cell types by pro-inflammatory cytokines, including interferon-g, TNF-a, and interleukin-1, as well as by bacterial products (e.g. lipopolysaccharide) and phorbol esters (Mantovani et al., 1997). In addition to regulating cell migration, adhesion molecules and their ligands also play an important role in cellular differentiation and development, tissue repair, signal transduction and cell activation (Butcher, 1991; Yurochko, 1997). Recently, it has been reported that upregulation of cell adhesion molecules induced by shear stress helps in the process of arteriogenesis. During arteriogenesis upregulation of cell adhesion molecules helps in the accumulation of monocytes around the proliferating arterioles and provide several cytokines and growth factors (Van Royen et al., 2001).
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Leukocyte recruitment is critical for host defense leading to clearance of initiating factor or infection. Failure to express cell adhesion molecules leads to various pathological disorders as it impairs the individual’s ability to mount an inflammatory response. For example, in the skin of patients with lepromatous leprosy there is lack of expression of ICAM-1 on endothelial cells, resulting in a failure to develop immune granulomata in response to Mycobacteruim leprae (Moncada et al., 1993). In addition, two disorders arise due to deficient expression of cell adhesion molecules viz. leukocyte adhesion deficiency-1 (LAD-1) and leukocyte adhesion deficiency-2 (LAD-2). LAD-1 an autosomal recessive disorder arising due to deficient expression of b2 integrins viz. leukocyte-function-associated antigen-1 (LFA-1), Mac-1, and p150,95. In these patients, most of the adhesion-dependent functions of leukocytes, including adherence to endothelium, neutrophil aggregation and chemotaxis, phagocytosis, and cytotoxicity mediated by neutrophils, NK cells and T lymphocytes are abnormal leading to recurrent bacterial and fungal infections and impaired wound healing. LAD-2 patients have mutations in genes encoding enzymes involved in fucose metabolism leading to an absence of sialyl-lewisX, the carbohydrate ligand on neutrophils required for the binding to E- and P-selectin on cytokine-activated endothelium (Abbas, 1994). Canscora deccusata (Gentianaceae), popularly known as Shankpushpi in India, is a herb and is used as a laxative and a tonic. Several medicinal properties have been attributed to it in the traditional medicine in India. The crude dried powder of the herb and its ethanolic extract has been reported to have anti-convulsant activity (Dikshit et al., 1972). The ethanolic extract of the herb lowers blood pressure in albino rats and also shows stimulant action on the smooth muscle of intestine, uterus and bronchus. It also has spermicidal activity in rats (Ramachandran, 1992). Purified xanthones from C. decussata have shown inhibitory activity against Mycobacterium tuberculosis H37RV (Ghosal et al., 1978). Although, alcoholic extracts of C. decussata have been studied but there are no reports so far on activity or characterization of its aqueous components. Also, no mechanism has been proposed to explain the reported activities of C. decussata. We hypothesize that some of the activities of C. decussata reported in the traditional medicine of India could be due its role in regulating cellular trafficking by regulating the expression of cell adhesion molecules. In this study, we show that an aqueous extract of C. decussata promotes the adhesion of neutrophils to endothelial cells by upregulating the expression of cell adhesion molecules on endothelial cells. We also show that this extract of C. decussata promotes the transcription of genes encoding these adhesion molecules.
2. Methodology
2.1. Materials Anti-ICAM-1 (BBA3) and -E-selectin (BBA1) antibodies were purchased from R&D Systems (Minneapolis, MN). M199, L-glutamine, penicillin, streptomycin, amphotericin, endothelial cell growth factor, trypsin, Pucks saline, HEPES, o-phenylenediamine dihydrochloride, ficoll-hypaque, cetitrimethyl ammonium bromide, 3-amino-1,2,4 triazole, anti-mouse IgGHRP were purchased from Sigma Chemical Co., USA. The ICAM-1, VCAM-1, E-selectin and b-actin primer sets employed were synthesized by Genset Corp. (Tokyo, Japan). Fetal calf serum was purchased from Biological industries (Kibbutz Beit Haemek, Israel).
2.2. Preparation of the extract Dried powdered rhizomes of C. decussata (1 g), were macerated in 100 ml of distilled water overnight at 25 °C under shaking and the percolate was collected. Maceration and percolate collection was continued for 2 days. The percolate was pooled and vacuum dried. The yield of this process was 7.0% (w/w). The dried material was resuspended in distilled water before use at a concentration of 2 mg/ml and is designated as CdAqE.
2.3. Cells and cell culture Endothelial cells were isolated from human umbilical cords by mild trypsinisation (Gupta and Ghosh, 1999). The cells were cultured in M199 medium supplemented with 20% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin and endothelial cell growth factor (50 mg/ml) in gelatin coated tissue culture flasks. For subculturing, the cells were dislodged using 0.125% trypsin–0.01M EDTA solution in Pucks saline and HEPES buffer. The cells were used in passages 3 and 4. The viability of cells was determined by trypan blue staining and their identity was established by E-selectin expression.
2.4. Neutrophil isolation Neutrophils were isolated from peripheral blood as described previously (Madan et al., 2000). Blood from healthy individuals was collected in heparin solution and the red blood cells were removed by sedimentation in 6% dextran. The white-blood-cell rich plasma layer was collected, and layered on a ficoll-hypaque solution and centrifuged for 20 min at 300× g and 20 °C. Following centrifugation, the top saline layer and the ficoll-hypaque layer were removed and the neutrophil/
B. Madan, B. Ghosh / Journal of Ethnopharmacology 79 (2002) 229–235
RBC pellet was collected. Residual RBC’s were removed by hypotonic lysis. The isolated cells were washed with PBS and resuspended in PBS containing 5 mM glucose, 1 mM CaCl2 and 1 mM MgCl2 to a concentration of 6× 105 cells/ml. This procedure usually results in approximately 95% pure neutrophils with a cell viability of more than 95% as detected by trypan blue exclusion.
2.5. Cell-adherence assay The adherence assay was performed as described before (Madan et al., 2000). To assay the adhesion of neutrophils to endothelial monolayers, the endothelial cells were plated to confluence in 96-well culture plates. The cells were incubated with or without CdAqE and/ or TNF-a (10 ng/ml) for 6 h. After incubation, the cells were washed two times with PBS and then incubated with neutrophils (6×104/well) for 1 h at 37 °C. The endothelial monolayers were washed with PBS to remove the non-adherent neutrophils. Adherent neutrophils were assayed colorimetrically by adding substrate solution (100 ml/well) consisting of peroxidase substrate (o-phenylenediamine dihydrochloride 40 mg/ 100 ml in citrate phosphate buffer, pH 4.5) containing 0.1% cetitrimethyl ammonium bromide and 3amino-1,2,4 triazole (1 mM). Reaction was stopped by adding 2 N H2SO4 (50 ml/well). The absorbance was determined at 490 nm using an automated microplate reader (Spectramax 190, Molecular Devices, USA).
2.6. Modified ELISA for the measurement of ICAM-1 and E-selectin The expression of ICAM-1 and E-selectin on the surface of endothelial cells was measured using cellELISA as described previously (Madan et al., 2001). Endothelial cells were grown to confluence in 96 well plates. The cells were incubated with or without CdAqE or with TNF-a (10 ng/ml) for 16 h for ICAM1 or for 4 h for E-selectin expression. Following incubation, the cells were washed with PBS and fixed with glutaraldehyde. After fixation, the cells were blocked with 3.0% non-fat dry milk, and then incubated with anti-ICAM-1 or -E-selectin monoclonal antibody overnight at 4 °C. The cells were then washed with PBS and incubated with peroxidase-conjugated goat anti-mouse antibody. Subsequently, the cells were washed with PBS and exposed to peroxidase substrate (o-phenylenediamine dihydrochloride in citrate phosphate buffer, pH 4.5). Reaction was stopped by adding 2 N H2SO4 (50 ml/well). Absorbance was measured at 490 nm.
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2.7. Total RNA isolation and re6erse-transcription polymerase chain reaction (RT-PCR) Endothelial cells were treated with or without CdAqE or TNF-a (10 ng/ml) for 4 h. Following this, the total RNA was isolated from endothelial cells according to a modified guanidium thiocyanate procedure (Chomczynski and Sacchi, 1987). Expression of the transcripts for ICAM-1, VCAM-1 and E-selectin was evaluated by RT-PCR to yield products of length 555, 533, and 479 bp, respectively as detailed before (Gupta and Ghosh, 1999). b-actin was amplified simultaneously, as a control, using identical conditions resulting in an amplified product of 661 bp. RT-PCR was performed as a single step reaction following manufacturer’s protocol (Access RT-PCR system, Promega, Madison). PCR products were analyzed by 1%-agarose gel electrophoresis.
3. Results
3.1. CdAqE is non-toxic to endothelial cells To determine any possible toxic effect of CdAqE on endothelial cells, the cells grown to confluence in 96well plates were incubated with varying concentrations of CdAqE for 24 h. Viability of the cells was determined by trypan-blue exclusion and their morphology was observed under microscope. It was observed that the time of incubation (upto 24 h) and the extract concentration used (20 mg/ml) in subsequent experiments did not affect either the viability or morphology of the endothelial cells (data not shown).
3.2. CdAqE promotes the adhesion of neutrophils to endothelial cells To study the effect of CdAqE on the adhesion of neutrophils to endothelial cells, the endothelial cells were treated with CdAqE at concentrations varying from 2.5 to 20 mg/ml whereafter the adhesion of neutrophils to endothelial monolayers was evaluated. As studied by cell-adherence and shown in Fig. 1, the adhesion of neutrophils to the unstimulated endothelial cells was low. However, when endothelial cells were incubated with CdAqE, there was a concentration-dependent increase in the adhesion of neutrophils to endothelial cells, with maximal adhesion at a concentration of 20 mg/ml. The level of adhesion was comparable to the adhesion induced by TNF-a. Moreover, there was no further increase in adhesion induced by CdAqE upon further treatment of endothelial cells with TNF-a (data not shown).
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3.3. CdAqE upregulates ICAM-1 and E-selectin expression on endothelial cells in a concentration-dependent manner The adhesion of neutrophils to endothelial cells requires the expression of cell adhesion molecules such as ICAM-1 and E-selectin. Therefore, we studied the effect of CdAqE on the expression of these molecules. As detected by ELISA, and shown in Fig. 2A and B, ICAM-1 and E-selectin were expressed at low levels on unstimulated endothelial cells. When endothelial cells were incubated with CdAqE, a concentration-dependent upregulation of ICAM-1 and E-selectin expression was observed with maximal expression at a concentration of 20 mg/ml. Maximal levels of ICAM-1 and E-selectin expression induced by CdAqE were comparable with those for TNF-induced expression.
CdAqE. The endothelial cells were incubated with 20 mg/ml CdAqE or induced with TNF-a for 4 h. As revealed by RT-PCR analysis and shown in Fig. 3, there were low levels of ICAM-1 mRNA and undetectable levels of E-selectin and VCAM-1 mRNA in unstimulated cells (lanes 1, 4 and 7). Treatment with CdAqE (20 mg/ml) significantly increased the transcript levels of ICAM-1, E-selectin and VCAM-1 (lanes 3, 6 and 9, respectively). The level of expression of ICAM-1, VCAM-1 and E-selectin in cells treated with CdAqE was similar to the level in TNF-a induced cells (lanes 2, 5 and 8). The levels of b-actin mRNA expressed under these conditions remained the same (lanes 10– 13). These results indicate that CdAqE upregulates the expression of ICAM-1 and E-selectin by activating transcription of their genes. In addition to ICAM-1 and E-selectin, CdAqE also induces the expression of VCAM-1 on endothelial cells.
3.4. Transcript le6els of ICAM-1, E-selectin and VCAM-1 are significantly increased by CdAqE 4. Discussion and conclusions In order to understand the mechanism of induction of cell adhesion molecules by CdAqE, we analyzed the steady-state transcript levels of ICAM-1 and E-selectin in the presence of CdAqE. In addition to ICAM-1 and E-selectin, VCAM-1 is also an important cell adhesion molecule required for the adhesion of eosinophils, lymphocytes, and monocytes to the endothelial cells. Therefore, we also analyzed the steady-state transcript levels of VCAM-1 in endothelial cells in the presence of
The cell adhesion molecules are important in the regulation of cellular immune responses as they control the extravasation and migration of body’s effector cells to the site of immune activation (Butcher, 1991). Failure of expression of these molecules impairs the immune system and leads to various pathological disorders. We demonstrate here for the first time that an aqueous preparation of a naturally and widely oc-
Fig. 1. Increase of neutrophil adhesion to endothelial cells. Endothelial cells grown to confluence in 96-well plates were incubated with or without indicated concentrations of CdAqE or with TNF-a (10 ng/ml) for 6 h. The cells were then incubated with human peripheral neutrophils for 1 h. The amounts of neutrophils adhering to the endothelial cell monolayers were measured by a colorimetric assay as described in Section 2. Data are representative of three independent experiments. Values shown are mean 9 SD of duplicate wells.
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Fig. 2. Concentration-dependent increase of ICAM-1 and E-selectin expression by CdAqE: (A) endothelial cells grown to confluence in 96-well plates were incubated with or without indicated concentrations of CdAqE or with TNF-a (10 ng/ml) for 16 h. ICAM-1 level on the cells was measured by ELISA as described in Section 2. (B) Endothelial cells were treated as in ‘A’ with or without CdAqE or with TNF-a (10 ng/ml) for 4 h. Level of E-selectin on the cells was measured by ELISA as described in Section 2. Data are representative of three independent experiments. Values shown are mean + SD of duplicate wells.
curring plant C. decussata, can be used for promoting the adhesion of peripheral neutrophils to human umbilical vein endothelial cells. CdAqE promotes the adhesion of neutrophils by inducing the expression of cell adhesion molecules ICAM-1 and E-selectin on endothelial cells. Our RT-PCR results demonstrate that CdAqE increases the steady state transcript levels of these adhesion molecules suggesting that it may be activating at an early stage of signaling event.
Although, several medicinal properties such as antibacterial, anti-convulsant activities have been attributed to C. decussata in the traditional medicine in India but not much experimental evidence is present (Ramachandran, 1992). Our results showing the activation of cell-adhesion molecules by the aqueous preparation of C. decussata may explain some of the underlying mechanisms of reported activities of C. decussata. To mount an immune response against invading pathogens
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Fig. 3. Analysis of ICAM-1, VCAM-1, and E-selectin transcript levels in cells treated with CdAqE: Endothelial cells were incubated with CdAqE (20 mg/ml) or induced with TNF-a (10 ng/ml) for 4 h. Total RNA of the cells was isolated and analyzed by RT-PCR as described in the Section 2. Lane M, marker fX174 HaeIII digest; lanes 1, 4 and 7, unstimulated cells; lanes 2, 5 and 8, cells stimulated with TNF-a; lanes 3, 6 and 9 cells treated with CdAqE; lanes 1 –3 represent ICAM-1; lanes 4 – 6 represent VCAM-1 and lanes 7 – 9 represent E-selectin; corresponding samples were analyzed for b-actin mRNA as control (lanes 10 –12).
it is required for the cells of the immune system to migrate to the site of infection. The antibacterial activity of the extract or its components could be due to its potential to upregulate the expression of cell adhesion molecules. The anti-convulsant activity of C. decussata extract be possibly explained by the role of cell adhesion molecules in transducing signals to allow the neurons to respond to alteration in the neurite behavior and synapse formation. Similarly, various other reported activities of C. decussata may be explained as cell adhesion molecules mediate various cellular interactions, regulate leukocyte trafficking, help in antigen recognition and potentiate overall immune function. An aqueous preparation of C. decussata may be useful in conditions where upregulation of cell adhesion molecules is required. Topical application of CdAqE may be useful for inducing the expression of ICAM-1 on endothelial cells where there is a lack of its expression, for example, for developing an immune granuloma in response to M. leprae in patients with lepromatous leprosy (Moncada et al., 1993). Also in the process of arteriogenesis where the upregulated expression of cell adhesion molecules aids in the formation of capillary network, the use of CdAqE may be useful (Van Royen et al., 2001). CdAqE may also be useful for the treatment of localized infections and in wound healing as it can promote the infiltration of neutrophils at that site. The aqueous extract of C. decussata may thus find new therapeutic applications in the future.
Acknowledgements We thank Dr K. Muralidhar, University of Delhi for
his advice and constructive criticisms. Council of Scientific and Industrial Research, India, and Department of Biotechnology, India supported this work. Babita Madan is a recipient of CSIR fellowships.
References Abbas, A.K., 1994. Immune-mediated tissue injury and disease. In: Abbas, A.K., Litchman, A.H., Pober, J.S. (Eds.), Cellular and Molecular Immunology. WB Saunders, Co., USA, pp. 417 –418. Butcher, E.C., 1991. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67, 1033 –1036. Chomczynski, P., Sacchi, N., 1987. Single step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction. Analytical Biochemistry 162, 156 – 159. Dikshit, S.K., Tewari, P.V., Dixit, S.P., 1972. Anticonvulsant activity of Canscora decussata Roem. and Sch. Indian Journal of Physiology and Pharmacology 16, 81 – 83. Ghosal, S., Biswas, K., Chaudhuri, R.K., 1978. Chemical constituents of Gentianaceae XXIV: anti-Mycobacterium tuberculosis activity of naturally occurring xanthones and synthetic analogs. Journal of Pharmaceutical Science 67, 721 – 722. Gupta, B., Ghosh, B., 1999. Curcuma longa inhibits TNF-a induced expression of adhesion molecules on human umbilical vein endothelial cells. International Journal of Immunopharmacology 21, 745 – 757. Madan, B., Batra, S., Ghosh, B., 2000. 2%-Hydroxychalcone inhibits nuclear factor-kB and blocks tumor necrosis factor-a- and lipopolysaccharide-induced adhesion of neutrophils to human umbilical vein endothelial cells. Molecular Pharmacology 58, 526 – 534. Madan, B., Gade, W., Ghosh, B., 2001. C. longa aqueous extract activates NF-kB and promotes the adhesion of neutrophils endothelial cells. Journal of Ethnopharmacology 75, 25 – 32. Mantovani, A., Bussolino, F., Introna, M., 1997. Cytokine regulation of endothelial cell function: from molecular level to bedside. Immunology Today 18, 231 – 240. Moncada, B., Torres-Alvarez, M.B., Gonzalez-Amaro, R., FuentesAhumada, C., Baranda, L., Delgado, S.P., Garcia, R., 1993.
B. Madan, B. Ghosh / Journal of Ethnopharmacology 79 (2002) 229–235 Lack of expression of intercellular adhesion molecule ICAM-1 in lepromatous leprosy patients. International Journal of Leprosy and Other Mycobacterial Diseases 6, 581 –585. Ramachandran, K., 1992. Wealth of India (Raw Materials). Publications and Information Directorate, Council of Scientific and Industrial Research, Delhi, 3: p. 205. Van Royen, N., Pick, J.J., Buschmann, I., Hoefer, I., Voskuil, M.,
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Schaper, W., 2001. Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease. Cardiovascular Research 49, 543 – 553. Yurochko, A.D., 1997. Adhesion molecules as signal transduction molecules. In: Paul, L.C., Issekutz, T.B. (Eds.), Adhesion Molecules in Health and Disease. Marcel Dekker Inc., New York, pp. 155 – 180.
Canscora decussata promotes adhesion of neutrophils to human umbilical vein endothelial cells Babita Madan, Balaram Ghosh * Molecular Immunology and Immunogenetics Laboratory, Centre for Biochemical Technology, Uni6ersity of Delhi Campus (North), Mall Road, Delhi 110 007, India Accepted 1 November 2001
Abstract A regulated expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells is essentially required for immune surveillance. Cell adhesion molecules regulate the extravasation and migration of body’s effector cells to the site of immune activation. Failure of expression of cell adhesion molecules leads to various pathological diseases as it impairs the individuals’ ability to mount an inflammatory response. Our results demonstrate that an aqueous extract prepared from Canscora decussata (CdAqE), promotes the adhesion of peripheral neutrophils to human umbilical vein endothelial cells. The increased adhesion is as a result of induction of the expression of ICAM-1 and E-selectin on endothelial cells. CdAqE increases the expression of steady state transcript levels of these molecules as determined by RT-PCR. These results have implications for the usage of aqueous preparation of C. decussata for upregulation of cell adhesion molecule expression. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Canscora decussata; Endothelial cells; Neutrophils; Adhesion molecules; Immunomodulators
1. Introduction Lymphocytes continually patrol the body for foreign antigens and allow efficient surveillance of the tissues. In response to any injury or infection the endothelium gets activated and the leukocytes are recruited locally into the site by various chemotactic agents including lipopolysaccharide derived from bacterial cell walls, cytokines, and eicosanoids produced by local tissue monocytes and endothelial cells. A regulated expression of cell adhesion molecules such as ICAM-1 (intercellu-
Abbre6iations: ICAM-1, intercellular adhesion molecule-1; VCAM1, vascular cell adhesion molecule-1; TNF-a, tumor necrosis factor-a; LPS, lipopolysaccharide; IL-1, interleukin-1; Mac-1, CD11b/CD18; HUVECs, human umbilical vein endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; mAb, monoclonal antibody; PBS, phosphate buffer saline. * Corresponding author. Tel.: +91-11-766-7602; fax: + 91-11-7667471. E-mail address: [email protected] (B. Ghosh).
lar adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), and E-selectin on the endothelium controls the trafficking of the lymphocytes and their migration to the sites of injury or infection. These cell adhesion molecules are not expressed on unstimulated endothelial cells but their expression is induced on various cell types by pro-inflammatory cytokines, including interferon-g, TNF-a, and interleukin-1, as well as by bacterial products (e.g. lipopolysaccharide) and phorbol esters (Mantovani et al., 1997). In addition to regulating cell migration, adhesion molecules and their ligands also play an important role in cellular differentiation and development, tissue repair, signal transduction and cell activation (Butcher, 1991; Yurochko, 1997). Recently, it has been reported that upregulation of cell adhesion molecules induced by shear stress helps in the process of arteriogenesis. During arteriogenesis upregulation of cell adhesion molecules helps in the accumulation of monocytes around the proliferating arterioles and provide several cytokines and growth factors (Van Royen et al., 2001).
0378-8741/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 3 8 6 - 5
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Leukocyte recruitment is critical for host defense leading to clearance of initiating factor or infection. Failure to express cell adhesion molecules leads to various pathological disorders as it impairs the individual’s ability to mount an inflammatory response. For example, in the skin of patients with lepromatous leprosy there is lack of expression of ICAM-1 on endothelial cells, resulting in a failure to develop immune granulomata in response to Mycobacteruim leprae (Moncada et al., 1993). In addition, two disorders arise due to deficient expression of cell adhesion molecules viz. leukocyte adhesion deficiency-1 (LAD-1) and leukocyte adhesion deficiency-2 (LAD-2). LAD-1 an autosomal recessive disorder arising due to deficient expression of b2 integrins viz. leukocyte-function-associated antigen-1 (LFA-1), Mac-1, and p150,95. In these patients, most of the adhesion-dependent functions of leukocytes, including adherence to endothelium, neutrophil aggregation and chemotaxis, phagocytosis, and cytotoxicity mediated by neutrophils, NK cells and T lymphocytes are abnormal leading to recurrent bacterial and fungal infections and impaired wound healing. LAD-2 patients have mutations in genes encoding enzymes involved in fucose metabolism leading to an absence of sialyl-lewisX, the carbohydrate ligand on neutrophils required for the binding to E- and P-selectin on cytokine-activated endothelium (Abbas, 1994). Canscora deccusata (Gentianaceae), popularly known as Shankpushpi in India, is a herb and is used as a laxative and a tonic. Several medicinal properties have been attributed to it in the traditional medicine in India. The crude dried powder of the herb and its ethanolic extract has been reported to have anti-convulsant activity (Dikshit et al., 1972). The ethanolic extract of the herb lowers blood pressure in albino rats and also shows stimulant action on the smooth muscle of intestine, uterus and bronchus. It also has spermicidal activity in rats (Ramachandran, 1992). Purified xanthones from C. decussata have shown inhibitory activity against Mycobacterium tuberculosis H37RV (Ghosal et al., 1978). Although, alcoholic extracts of C. decussata have been studied but there are no reports so far on activity or characterization of its aqueous components. Also, no mechanism has been proposed to explain the reported activities of C. decussata. We hypothesize that some of the activities of C. decussata reported in the traditional medicine of India could be due its role in regulating cellular trafficking by regulating the expression of cell adhesion molecules. In this study, we show that an aqueous extract of C. decussata promotes the adhesion of neutrophils to endothelial cells by upregulating the expression of cell adhesion molecules on endothelial cells. We also show that this extract of C. decussata promotes the transcription of genes encoding these adhesion molecules.
2. Methodology
2.1. Materials Anti-ICAM-1 (BBA3) and -E-selectin (BBA1) antibodies were purchased from R&D Systems (Minneapolis, MN). M199, L-glutamine, penicillin, streptomycin, amphotericin, endothelial cell growth factor, trypsin, Pucks saline, HEPES, o-phenylenediamine dihydrochloride, ficoll-hypaque, cetitrimethyl ammonium bromide, 3-amino-1,2,4 triazole, anti-mouse IgGHRP were purchased from Sigma Chemical Co., USA. The ICAM-1, VCAM-1, E-selectin and b-actin primer sets employed were synthesized by Genset Corp. (Tokyo, Japan). Fetal calf serum was purchased from Biological industries (Kibbutz Beit Haemek, Israel).
2.2. Preparation of the extract Dried powdered rhizomes of C. decussata (1 g), were macerated in 100 ml of distilled water overnight at 25 °C under shaking and the percolate was collected. Maceration and percolate collection was continued for 2 days. The percolate was pooled and vacuum dried. The yield of this process was 7.0% (w/w). The dried material was resuspended in distilled water before use at a concentration of 2 mg/ml and is designated as CdAqE.
2.3. Cells and cell culture Endothelial cells were isolated from human umbilical cords by mild trypsinisation (Gupta and Ghosh, 1999). The cells were cultured in M199 medium supplemented with 20% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin and endothelial cell growth factor (50 mg/ml) in gelatin coated tissue culture flasks. For subculturing, the cells were dislodged using 0.125% trypsin–0.01M EDTA solution in Pucks saline and HEPES buffer. The cells were used in passages 3 and 4. The viability of cells was determined by trypan blue staining and their identity was established by E-selectin expression.
2.4. Neutrophil isolation Neutrophils were isolated from peripheral blood as described previously (Madan et al., 2000). Blood from healthy individuals was collected in heparin solution and the red blood cells were removed by sedimentation in 6% dextran. The white-blood-cell rich plasma layer was collected, and layered on a ficoll-hypaque solution and centrifuged for 20 min at 300× g and 20 °C. Following centrifugation, the top saline layer and the ficoll-hypaque layer were removed and the neutrophil/
B. Madan, B. Ghosh / Journal of Ethnopharmacology 79 (2002) 229–235
RBC pellet was collected. Residual RBC’s were removed by hypotonic lysis. The isolated cells were washed with PBS and resuspended in PBS containing 5 mM glucose, 1 mM CaCl2 and 1 mM MgCl2 to a concentration of 6× 105 cells/ml. This procedure usually results in approximately 95% pure neutrophils with a cell viability of more than 95% as detected by trypan blue exclusion.
2.5. Cell-adherence assay The adherence assay was performed as described before (Madan et al., 2000). To assay the adhesion of neutrophils to endothelial monolayers, the endothelial cells were plated to confluence in 96-well culture plates. The cells were incubated with or without CdAqE and/ or TNF-a (10 ng/ml) for 6 h. After incubation, the cells were washed two times with PBS and then incubated with neutrophils (6×104/well) for 1 h at 37 °C. The endothelial monolayers were washed with PBS to remove the non-adherent neutrophils. Adherent neutrophils were assayed colorimetrically by adding substrate solution (100 ml/well) consisting of peroxidase substrate (o-phenylenediamine dihydrochloride 40 mg/ 100 ml in citrate phosphate buffer, pH 4.5) containing 0.1% cetitrimethyl ammonium bromide and 3amino-1,2,4 triazole (1 mM). Reaction was stopped by adding 2 N H2SO4 (50 ml/well). The absorbance was determined at 490 nm using an automated microplate reader (Spectramax 190, Molecular Devices, USA).
2.6. Modified ELISA for the measurement of ICAM-1 and E-selectin The expression of ICAM-1 and E-selectin on the surface of endothelial cells was measured using cellELISA as described previously (Madan et al., 2001). Endothelial cells were grown to confluence in 96 well plates. The cells were incubated with or without CdAqE or with TNF-a (10 ng/ml) for 16 h for ICAM1 or for 4 h for E-selectin expression. Following incubation, the cells were washed with PBS and fixed with glutaraldehyde. After fixation, the cells were blocked with 3.0% non-fat dry milk, and then incubated with anti-ICAM-1 or -E-selectin monoclonal antibody overnight at 4 °C. The cells were then washed with PBS and incubated with peroxidase-conjugated goat anti-mouse antibody. Subsequently, the cells were washed with PBS and exposed to peroxidase substrate (o-phenylenediamine dihydrochloride in citrate phosphate buffer, pH 4.5). Reaction was stopped by adding 2 N H2SO4 (50 ml/well). Absorbance was measured at 490 nm.
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2.7. Total RNA isolation and re6erse-transcription polymerase chain reaction (RT-PCR) Endothelial cells were treated with or without CdAqE or TNF-a (10 ng/ml) for 4 h. Following this, the total RNA was isolated from endothelial cells according to a modified guanidium thiocyanate procedure (Chomczynski and Sacchi, 1987). Expression of the transcripts for ICAM-1, VCAM-1 and E-selectin was evaluated by RT-PCR to yield products of length 555, 533, and 479 bp, respectively as detailed before (Gupta and Ghosh, 1999). b-actin was amplified simultaneously, as a control, using identical conditions resulting in an amplified product of 661 bp. RT-PCR was performed as a single step reaction following manufacturer’s protocol (Access RT-PCR system, Promega, Madison). PCR products were analyzed by 1%-agarose gel electrophoresis.
3. Results
3.1. CdAqE is non-toxic to endothelial cells To determine any possible toxic effect of CdAqE on endothelial cells, the cells grown to confluence in 96well plates were incubated with varying concentrations of CdAqE for 24 h. Viability of the cells was determined by trypan-blue exclusion and their morphology was observed under microscope. It was observed that the time of incubation (upto 24 h) and the extract concentration used (20 mg/ml) in subsequent experiments did not affect either the viability or morphology of the endothelial cells (data not shown).
3.2. CdAqE promotes the adhesion of neutrophils to endothelial cells To study the effect of CdAqE on the adhesion of neutrophils to endothelial cells, the endothelial cells were treated with CdAqE at concentrations varying from 2.5 to 20 mg/ml whereafter the adhesion of neutrophils to endothelial monolayers was evaluated. As studied by cell-adherence and shown in Fig. 1, the adhesion of neutrophils to the unstimulated endothelial cells was low. However, when endothelial cells were incubated with CdAqE, there was a concentration-dependent increase in the adhesion of neutrophils to endothelial cells, with maximal adhesion at a concentration of 20 mg/ml. The level of adhesion was comparable to the adhesion induced by TNF-a. Moreover, there was no further increase in adhesion induced by CdAqE upon further treatment of endothelial cells with TNF-a (data not shown).
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3.3. CdAqE upregulates ICAM-1 and E-selectin expression on endothelial cells in a concentration-dependent manner The adhesion of neutrophils to endothelial cells requires the expression of cell adhesion molecules such as ICAM-1 and E-selectin. Therefore, we studied the effect of CdAqE on the expression of these molecules. As detected by ELISA, and shown in Fig. 2A and B, ICAM-1 and E-selectin were expressed at low levels on unstimulated endothelial cells. When endothelial cells were incubated with CdAqE, a concentration-dependent upregulation of ICAM-1 and E-selectin expression was observed with maximal expression at a concentration of 20 mg/ml. Maximal levels of ICAM-1 and E-selectin expression induced by CdAqE were comparable with those for TNF-induced expression.
CdAqE. The endothelial cells were incubated with 20 mg/ml CdAqE or induced with TNF-a for 4 h. As revealed by RT-PCR analysis and shown in Fig. 3, there were low levels of ICAM-1 mRNA and undetectable levels of E-selectin and VCAM-1 mRNA in unstimulated cells (lanes 1, 4 and 7). Treatment with CdAqE (20 mg/ml) significantly increased the transcript levels of ICAM-1, E-selectin and VCAM-1 (lanes 3, 6 and 9, respectively). The level of expression of ICAM-1, VCAM-1 and E-selectin in cells treated with CdAqE was similar to the level in TNF-a induced cells (lanes 2, 5 and 8). The levels of b-actin mRNA expressed under these conditions remained the same (lanes 10– 13). These results indicate that CdAqE upregulates the expression of ICAM-1 and E-selectin by activating transcription of their genes. In addition to ICAM-1 and E-selectin, CdAqE also induces the expression of VCAM-1 on endothelial cells.
3.4. Transcript le6els of ICAM-1, E-selectin and VCAM-1 are significantly increased by CdAqE 4. Discussion and conclusions In order to understand the mechanism of induction of cell adhesion molecules by CdAqE, we analyzed the steady-state transcript levels of ICAM-1 and E-selectin in the presence of CdAqE. In addition to ICAM-1 and E-selectin, VCAM-1 is also an important cell adhesion molecule required for the adhesion of eosinophils, lymphocytes, and monocytes to the endothelial cells. Therefore, we also analyzed the steady-state transcript levels of VCAM-1 in endothelial cells in the presence of
The cell adhesion molecules are important in the regulation of cellular immune responses as they control the extravasation and migration of body’s effector cells to the site of immune activation (Butcher, 1991). Failure of expression of these molecules impairs the immune system and leads to various pathological disorders. We demonstrate here for the first time that an aqueous preparation of a naturally and widely oc-
Fig. 1. Increase of neutrophil adhesion to endothelial cells. Endothelial cells grown to confluence in 96-well plates were incubated with or without indicated concentrations of CdAqE or with TNF-a (10 ng/ml) for 6 h. The cells were then incubated with human peripheral neutrophils for 1 h. The amounts of neutrophils adhering to the endothelial cell monolayers were measured by a colorimetric assay as described in Section 2. Data are representative of three independent experiments. Values shown are mean 9 SD of duplicate wells.
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Fig. 2. Concentration-dependent increase of ICAM-1 and E-selectin expression by CdAqE: (A) endothelial cells grown to confluence in 96-well plates were incubated with or without indicated concentrations of CdAqE or with TNF-a (10 ng/ml) for 16 h. ICAM-1 level on the cells was measured by ELISA as described in Section 2. (B) Endothelial cells were treated as in ‘A’ with or without CdAqE or with TNF-a (10 ng/ml) for 4 h. Level of E-selectin on the cells was measured by ELISA as described in Section 2. Data are representative of three independent experiments. Values shown are mean + SD of duplicate wells.
curring plant C. decussata, can be used for promoting the adhesion of peripheral neutrophils to human umbilical vein endothelial cells. CdAqE promotes the adhesion of neutrophils by inducing the expression of cell adhesion molecules ICAM-1 and E-selectin on endothelial cells. Our RT-PCR results demonstrate that CdAqE increases the steady state transcript levels of these adhesion molecules suggesting that it may be activating at an early stage of signaling event.
Although, several medicinal properties such as antibacterial, anti-convulsant activities have been attributed to C. decussata in the traditional medicine in India but not much experimental evidence is present (Ramachandran, 1992). Our results showing the activation of cell-adhesion molecules by the aqueous preparation of C. decussata may explain some of the underlying mechanisms of reported activities of C. decussata. To mount an immune response against invading pathogens
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Fig. 3. Analysis of ICAM-1, VCAM-1, and E-selectin transcript levels in cells treated with CdAqE: Endothelial cells were incubated with CdAqE (20 mg/ml) or induced with TNF-a (10 ng/ml) for 4 h. Total RNA of the cells was isolated and analyzed by RT-PCR as described in the Section 2. Lane M, marker fX174 HaeIII digest; lanes 1, 4 and 7, unstimulated cells; lanes 2, 5 and 8, cells stimulated with TNF-a; lanes 3, 6 and 9 cells treated with CdAqE; lanes 1 –3 represent ICAM-1; lanes 4 – 6 represent VCAM-1 and lanes 7 – 9 represent E-selectin; corresponding samples were analyzed for b-actin mRNA as control (lanes 10 –12).
it is required for the cells of the immune system to migrate to the site of infection. The antibacterial activity of the extract or its components could be due to its potential to upregulate the expression of cell adhesion molecules. The anti-convulsant activity of C. decussata extract be possibly explained by the role of cell adhesion molecules in transducing signals to allow the neurons to respond to alteration in the neurite behavior and synapse formation. Similarly, various other reported activities of C. decussata may be explained as cell adhesion molecules mediate various cellular interactions, regulate leukocyte trafficking, help in antigen recognition and potentiate overall immune function. An aqueous preparation of C. decussata may be useful in conditions where upregulation of cell adhesion molecules is required. Topical application of CdAqE may be useful for inducing the expression of ICAM-1 on endothelial cells where there is a lack of its expression, for example, for developing an immune granuloma in response to M. leprae in patients with lepromatous leprosy (Moncada et al., 1993). Also in the process of arteriogenesis where the upregulated expression of cell adhesion molecules aids in the formation of capillary network, the use of CdAqE may be useful (Van Royen et al., 2001). CdAqE may also be useful for the treatment of localized infections and in wound healing as it can promote the infiltration of neutrophils at that site. The aqueous extract of C. decussata may thus find new therapeutic applications in the future.
Acknowledgements We thank Dr K. Muralidhar, University of Delhi for
his advice and constructive criticisms. Council of Scientific and Industrial Research, India, and Department of Biotechnology, India supported this work. Babita Madan is a recipient of CSIR fellowships.
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