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Cell Biology International (2003) 27, 375382 (Printed in Great Britain)
Targeting apoptotic signalling pathway and pro-inflammatory cytokine expression as therapeutic intervention in TPE induced lung damage
Kishore Narayanana, Bhavani Krishnamoorthya, Ravesanker Ezhilarasana, Shigeki Miyamotob and Arun Balakrishnana*
aCentre for Biotechnology, Anna University, Chennai 600025, India
bDepartment of Pharmacology, University of Wisconsin, Madison, WI 53792, USA
Tropical pulmonary eosinophilia (TPE) is an occult manifestation of filariasis, brought about by helminth parasites Wuchereria bancrofti and Brugia malayi. Treatment of patients suffering from TPE involves the administration of diethyl carbamazine and Ivermectin. Although the drugs are able to block acute inflammation, they are not able to alleviate chronic basal inflammation. We have attempted to examine the disease by targeting two important components; namely filarial parasitic sheath proteins (FPP) induced apoptosis and pro-inflammatory cytokine response in human laryngeal carcinoma cells of epithelial origin (HEp-2) cells an epithelial cell line. Earlier studies by us have shown that FPP exposure induced apoptosis in these cells. In this study with hydrocortisone, calpain inhibitor (ALLN) and phorbol myristate acetate (PMA) treatments we demonstrate that apoptosis is inhibited as shown by [3H] thymidine incorporation studies, propidium iodide staining and Annexin V staining. Hydrocortisone at a dose, which inhibits cell death also down regulated, the expression of pro-inflammatory cytokines IL-6 and IL-8. These findings give us insights into the multifaceted approach one may adopt to target critical signalling molecules using appropriate inhibitors, which could eventually be used to reduce lung damage in TPE.
Keywords: Inflammation, Lung-epithelium, Apoptosis, Inhibitors and lung damage.
*Corresponding author. Tel.: +91-44-235-0299; fax: +91-44-235-0299.
Tropical pulmonary eosinophilia (TPE), an occult form of filariasis, is an interstitial lung disease due to an exaggerated immune response to parasites Wuchereria bancrofti and Brugia malayi (Barnes and Karin, 1997). Acute TPE is characterised by an intense inflammatory process in the lower respiratory tract (Ong and Doyle, 1998; Ottesen and Nutman, 1992). Investigations have expanded our understanding of the immune mechanisms to different degrees of worm burden (Ottesen and Nutman, 1992) parasite mediated damage in the lung (Ueda et al., 1996), the elevation of IgG4 and IgE levels (Ottesen and Nutman, 1992) and the localisation of eosinophils in the lung (Pinkston et al., 1987). In TPE, extensive damage to the lung epithelium occurs due to an exaggerated immune response, characterised by an intense eosinophilic alveolitis. However, not much is known as to how such an inflammation is mediated. The question thus arises as to whether the immune responsiveness that is indeed observed is a primary event, which then causes damage to the lung, or is a secondary event that culminates as a result of the stimulus released by dying epithelial cells.
A large body of evidence is directed towards the role of the epithelial cells in mediating inflammatory response by secretion of molecular signals such as cytokines and chemokines (Levine, 1995). The lung epithelium poses as the first physical barrier to most physical and biological insults and therefore it plays a critical role in orchestrating the local immune response against invading pathogens (Leff et al., 1991). Increased levels of pro-inflammatory cytokines are seen in the lung epithelial lining fluid in several pathogenic conditions (Lazarus, 1986). Under in vitro conditions epithelial cells respond to Escherichia coli proteins by producing IL-6, IL-8 and IL-1β (Jung et al., 1995).
In the earlier work reported by us, we have shown the use of human laryngeal carcinoma cells of epithelial origin (HEp-2) cell lines to understand the molecular mechanisms involved during filarial parasitic sheath proteins (FPP) induced apoptosis. Although these are laryngio carcinoma cell lines of epithelial origin, using these well-characterised cell lines, the possible effect of FPP has been identified. Similar studies are presently being done on lung derived epithelial cell lines.
In this paper we have analysed the possible anti-apoptotic and anti-inflammatory effects of drugs like hydrocortisone, calpain inhibitor (ALLN) and phorbol myristate acetate (PMA) that specifically target two important pathways that lead to cell death namely the apoptotic pathway and the inflammatory reaction that culminates as a result of a direct interaction with FPP. Hydrocortisone is a known anti-inflammatory drug and controls inflammatory response by down regulating the expression of pro-inflammatory cytokines, but also blocks apoptosis mediated by TNF-α and LPS (Messmer et al., 1999, 2000). Calpain inhibitor ALLN blocks a group of enzymes called calpains. They play a critical role in preventing apoptotic cell death mediated by the loss of contact of the cell with the extracellular matrix (Squier et al., 1994; Stracher, 1999). Phorbol esters (PMA) modulate apoptosis by manipulating PKC and have been widely implicated in induction and suppression of apoptosis (Deacon et al., 1997). Long-term PMA treatment of cells results in inhibition of PKC due to the down regulation of PMA sensitive PKC isoforms (Vrana et al., 1999). The results in the present study indicate that hydrocortisone, PMA and ALLN block apoptosis vide acting through their specific cascades and down regulating FPP induced apoptosis. We feel that in such conditions of a chronic inflammatory disease of the lung, localised inhibition of such targets may prove to be valuable in reducing lung damage.
2 Materials and methods
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS) and Penicillin and Streptomycin (250 units/ml) were obtained from Life Technologies, USA (GIBCO BRL). [3H]-thymidine was obtained from Amersham, UK. Propidium iodide and Annexin V staining kit were obtained from Pharmingen International, UK. A saline solubilised extract of Setaria digitata was prepared as described earlier (Maya et al., 1997) and used at a dose of 62.5
2.2 Cell culture
HEp-2 (ATCC CCL-23) were obtained from (ATCC-USA). Passaged once in media provided by the manufacturer and for further use cells were maintained in DMEM with 10% FBS with antibiotics, in 100
2.3 Reversal of inhibition of cell proliferation during prolonged exposure to FPP by hydrocortisone, ALLN and PMA
The effect of FPP on HEp-2 cells was monitored by [3H]-thymidine incorporation (1
HEp-2 cells exposed to increasing doses of hydrocortisone (0.005, 0.025, 0.0125 and 0.05
2.4 Inhibition of apoptosis induced by FPP in the presence of hydrocortisone, ALLN and PMA (100 ng) detected by Annexin V and propidium iodide staining
HEp-2 cells exposed to different doses of hydrocortisone (0.005, 0.025, 0.0125 and 0.05
HEp-2 cells exposed to the inhibitor and FPP for the required time were harvested and washed twice with ice cold PBS. One set of cells was then resuspended in 1× binding buffer (10
Another set of cells was then fixed in 70% ethanol for half an hour at −20
2.5 RT-PCR for IL-6 and IL-8 expression in HEp-2 cells exposed to FPP in the presence and absence of hydrocortisone
RT-PCR was carried out as described in Hall et al. (1998). Briefly, cells exposed to FPP and LPS for 16 and 4
In order to evaluate the possible anti-inflammatory role of hydrocortisone in inhibiting the expression of pro-inflammatory cytokines like IL-6 and IL-8, total RNA was isolated from HEp-2 cells treated with FPP and exposed to with or without 0.0125
3.1 Hydrocortisone blocks FPP mediated cell death
In order to evaluate the role of hydrocortisone to block cell death, a dose response study with increasing doses of hydrocortisone ranging from 0.005 to 0.05
Hydrocortisone inhibits FPP mediated cell death as observed by [3H] thymidine incorporation studies. HEp-2 cells were treated with increasing doses of hydrocortisone (0.005, 0.025, 0.0125, and 0.05
3.2 Calpain inhibitor ALLN blocks FPP mediated cell death
A dose response study was carried out using increasing doses of ALLN (10, 50 and 100
ALLN inhibits FPP mediated cell death as observed by [3H] thymidine incorporation studies. HEp-2 cells were treated with increasing doses of ALLN (10, 50 and 100
3.3 PMA enhances protein kinase induced apoptosis at 50 ng/ml and suppresses apoptosis at 100 ng/ml
A dose response analysis with different doses of PMA was done and the and the doses of 50 and 100
PMA enhances survival of HEp-2 cells on exposure to 50 and 100
Propidium iodide and Annexin V staining of HEp-2 cells treated with FPP in the presence and absence of hydrocortisone, ALLN and PMA. Control HEp-2 cells show no apoptotic bodies (A,C) and cells treated with FPP show formation of typical apoptotic bodies (B,D). HEp-2 cells exposed to 0.0125
3.4 Hydrocortisone inhibits expression of pro-inflammatory cytokines IL-6 and IL-8 in HEp-2 cells on exposure to FPP
In order to evaluate the role of hydrocortisone in regulating the expression of pro-inflammatory cytokines, we analysed the expression of cytokines IL-6 and IL-8. Under the experimental conditions reported above, the expression of IL-6 and IL-8 were observed in HEp-2 cells exposed to FPP (Fig. 5, FPP) and absent in untreated controls (Fig. 5, control). A partial inhibition in the expression of IL-6 and IL-8 was observed in cells exposed to FPP and treated with 0.0125
RT-PCR analysis of pro-inflammatory cytokine transcripts for IL-6 and IL-8 in HEp-2 cells in the presence and absence of FPP and hydrocortisone. MW indicates molecular weight markers. Control indicates untreated HEp-2 cells; FPP indicates HEp-2 cells treated with sheath proteins; HC indicates HEp-2 cells treated with hydrocortisone (0.0125
Expression of IL-6 and IL-8 are elevated when the cells are treated with FPP. When cells were treated with hydrocortisone alone, which is an anti-inflammatory drug, no elevation in the levels of IL-6 and IL-8 were noticed and this was similar in untreated controls. However, in the state of increased expression of IL-6 and IL-8 on exposure to FPP, addition of hydrocortisone partially suppressed the expression of IL-6 and IL-8.
Development of a strategy to alleviate an inflammatory disease requires the understanding of the signalling events leading to the disease. In TPE there is extensive damage of the alveolar epithelium by the sheath proteins released by the degenerating microfilariae. Earlier work from our laboratory, which was directed towards understanding the signalling events that are associated with the disease, revealed that the direct interaction of FPP with epithelial cells leads to cell death by apoptosis; molecular effectors of the apoptotic pathway like cmyc and PKC were elevated (Maya et al., 1997); the over expression of bcl2 in HEp2 cells inhibited apoptosis (Krishnamoorthy et al., 1998) and also the expression of pro-inflammatory cytokines like IL-6 and IL-8 both at the m-RNA and the protein level (Krishnamoorthy et al., 2000). Heat treatment (90
Though many of the above studies have gone into evaluating the pathology of the disease, little has been done towards identifying a therapeutic strategy for reducing tissue destruction in TPE or other inflammatory conditions. The treatment of patients suffering from TPE, presently involves the use of diethyl carbamazine (Rom et al., 1990; Von Asmuth et al., 1994). Though it alleviates acute inflammation, it does not help to cure the patient of chronic inflammation. We have targeted two important aspects of the disease, one being the inflammatory response of the epithelium towards the shed sheath proteins and the other being the cell death of epithelium on exposure to FPP that leads to lung destruction. Hydrocortisone has been shown to prevent airway inflammation by down regulating the synthesis and release of pro-inflammatory mediators from bronchial epithelial cells (Trapp et al., 1998). Hydrocortisone is able to inhibit expression of a variety of pro-inflammatory cytokines, which include GM-CSF, TNF-α, IL-8 and IL-6 in human bronchial epithelial cells exposed to inhaled insults (Kato and shcleimer 1994; Khair et al., 1994). Hydrocortisone at a concentration of 10−5M (4
One of the possible ways to reduce damage to the epithelial cells, during an acute inflammatory stress, is to target and inhibit cellular cascades that are directly implicated in apoptosis. In this regard PMA has been considered as a drug in cancers where at specific doses they enhance apoptosis (Nishio and Watanabe, 1997; Ong and Doyle, 1998). PMA also plays a dual role on the basis of the concentration and/or the time of exposure (Ueda et al., 1996) as observed in retinoic acid-induced apoptosis of neuronal cells, where chronic stimulation with PMA may lead to down regulation of PKC and significantly reduce PKC-induced apoptosis (Mailhos et al., 1994).
Our studies on the effect of phorbol ester, PMA suggest that it activates PKC mediated apoptosis (Krishnamoorthy et al., 2000) at 50
During an inflammatory stress, apoptosis is also induced by the loss of epithelial–extra cellular matrix interactions, which is regulated by a set of proteases. Calpain is a protease, which is known to induce apoptosis by cleaving extracellular matrix–cellular interactions and activating important proteases like caspases in the apoptotic cascade (Kohli et al., 1999; Squier et al., 1994), which is blocked specifically by calpain inhibitors (Gressner et al., 1997; Stracher, 1999). ALLN belongs to a family of aldehyde inhibitors that are known to inhibit apoptosis mediated by the loss of epithelial–extracellular matrix interactions (Nath et al., 1996). Our studies with ALLN in FPP induced apoptosis, has shown that ALLN was capable of recovering upto 50% of cell death as observed in the [3H] thymidine data (Fig. 2) and apoptotic bodies were considerably reduced (Fig. 4J). This further suggests that apoptosis triggered by FPP could be as a result of the loss of the extracellular matrix interactions with the epithelium. However, the mechanistic reasons for such an inhibition of cell death in relation to calpain is not known and is being pursued. Further studies regarding the specific mechanisms involved and targeted by these inhibitors would be very interesting and fruitful in the quest for finding new strategies to reduce lung damage in TPE.
In conclusion we have made an effort to device a multifaceted approach to first dismantle the complex signalling events that take place and to target critical signalling molecules using appropriate inhibitors. The study provides positive interludes to the multidisciplinary approaches one has to adopt to develop a strategy to block destruction of lung tissue in TPE.
We are grateful to Professor P. Kaliraj, Director, Centre for Biotechnology, Anna University, Chennai, for his support and encouragement. We would like to acknowledge CSIR, New Delhi for supporting B.K. and K.N. through the CSIR-SRF fellowship scheme and Lady Tata Memorial Trust, Bombay for supporting R.E. through the JRF scheme, during the period of their research work.
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Received 19 March 2002/22 October 2002; accepted 6 December 2002doi:10.1016/S1065-6995(03)00014-3