S Soluble Intercellular adhesion molecule (sICAM)

The scrum levels in atopic patients (296 ± 62 ng/ml) showed no difference from the normal levels established by the manufactures of the ELISA kit used (210.6ng/ml) and there was no change after treatment, 250.2 ± 20.1 ng/ml (Fig 6.2.4.8a).

B c S < U 1600 1 4 0 0 1200 1000 800 600 4 0 0 200 0 0 W EEKS

F ig u re 6.2.4.8a Soluble intercellular cellular adhesion molecule (slCAM )values in the serum of patients before and after treatment with ZPT. There was no significant change and the levels in the serum did not differ from levels found in controls.

6.3 DISCUSSION

In data presented previously ZPT has been shown to inhibit CD23 expression on monocytes and induce XL-10 and TNF-a. This current study now reveals immunological changes in patients undergoing treatment with this preparation. The parameters that were significantly altered by the in vivo treatment were levels of IgE complexes, slL-2 receptor, sVCAM and the inducibility of CD23.

There was a significant decrease in IgE complexes after treatment. In this research the nature of these complexes was not analysed but it would not be difficult to speculate that these were IgE complexed to anti IgE antibody which have previously been described ( Nawata et al., 1985 ; Quinti et al., 1986;Marone et al., 1989). Czech et al., 1995 showed no change in the IgE complexes after treatment of AE patients. However this group analysed these complexes after 2-3 weeks of treatment and suggested that this might have been too short a duration to observe a change in the IgG anti IgE antibodies as the half life of IgG antibodies is about 3 weeks. This decrease in complexes detected could lead to less complexed IgE binding to mast cells, B cells, eosinophils and monocytes through their IgE receptors and therefore a diminution in mediator production which is known to exacerbate skin damage. Patients being treated with ZPT did not show any reduction in total IgE in spite of a significant reduction in the extent of their eczema as judged by scoring of erythema and surface damage. Other studies in patients with AE undergoing treatment with corticosteroids, azathioprine (Johansson & Juhlin 1970), cyclosporin A (Munro et al., 1991) and even IFN- y ( Weindel et al., 1994) also failed to show an immediate fall in total serum IgE levels in spite of clinical improvement.

Another prominent feature in AE is the upregulation of the CD23 on PBMCs and Langerhans cells in the skin of patients with AE ( Melewicz et al 1981; Nakamura et al., 1981; Buckley et al ., 1992 ). In particular, the levels of CD23 on monocytes have correlated with superoxide production ( Polla et al., 1992) and induction of nitric oxide ( Mossalayi et al., 1994). In this study the results have shown two different effects of treatment on CD23 expression on peripheral blood monocytes. Firstly, the percentage of circulating monocytes expressing CD23 following treatment was unchanged, although the expression of CD23 on monocytes in this group of patients was much lower than previously described ( Nakamura et al., 1991). Secondly, the level of lL-4 induced CD23 expression on monocytes in vitro was significantly reduced after treatment. This indicates that treatment is able to block lL-4

induction of CD23. This is in accordance with the finding that ZPT but not PL, can inhibit IL-4 induced CD23 expression in vitro. The in vivo treatment with ZPT did not effect its in vitro inhibition which may indicate that this treatment is inducing factors like cytokines which influence the immune system rather than altering the monocytes per se. Although CD23 on blood monocytes and sCD23 in the serum were not affected, biopsy data in other studies with this treatment have shown that CD23 is downregulated on antigen presenting cells in the skin of treated patients (Xiou et al submitted for publication ). This might suggest that this therapy is directed at the site of inflammation where the cells are activated rather than acting as a general immunosuppressant.

Activated T cells express the IL-2 R, of which the a chain can be shed and maintain its affinity for IL-2 (Rubin et al., 1986). This soluble form (sIL-2R) can be detected in the serum of AE patients. The level of sIL-2R correlates with disease activity and is reduced after treatment with corticosteroids (Colver et al., 1989, Wuthrich et al., 1990). The results presented do show a small but significant decrease in sIL-2R in the serum of patients on treatment. Shed IL-2R is a reflection of surface expression thus a reduction in this marker may mirror a decrease in activated T cells either as a primary or secondary phenomenon.

For the first time it has been shown that sVCAM levels are significantly raised in patients with AE compared with controls. There is also a reduction in sVCAM after 8 weeks of treatment. As VCAM is an adhesion molecule which preferentially binds basophils and eosinophils and is induced by IL-4, it may have a potential role in allergic reactions (Schleimer et al., 1992). The relationship between the expression of VCAM and the soluble form is not known but the decrease in sVCAM in the serum could be a reflection of downregulation of adhesion molecules on the endothelial cells and therefore a reduction in cells entering the skin. sICAM has recently been implicated as a marker for AE with levels decreasing on treatment with topical steroids ( Kojima et a l ., 1994; Wuthrich et al., 1995 ). In this study no difference was seen between sICAM levels in controls and patients and there was no change following treatment. Wuthrich et al., 1995 has suggested that ICAM may be a new clinical marker for AE, however our results do not support that conclusion. These data suggest that sVCAM may be more relevant as a diagnostic marker. To justify the relevance of our observations it will be necessary to show that VCAM and IL-2R are downregulated in the skin biopsies of AE patients after treatment.

This study has confirmed the efficacy of ZPT in patients with AE who have been unresponsive to standard Western Medicine. The treatment consists of a mixture of ten herbs of which many of the components may be unnecessary . The final aim must be the isolation of a compound or compounds which are responsible for the in vivo efficacy.

Chapter 7

Isolation and purification of factor(s) which inhibit CD23 expression on monocytes

7.1 INTRODUCTION

Two compounds used in treatment of atopic diseases, theophylline and disodium chromoglycate originated from plants. Theophylline, from the plant Theo sinensis belongs to the family of xanthines and is known for its ability to inhibit the PDE action (Butcher & Sutherland 1962). Its property as a bronchodiator has lead to its use in asthma. Patients with AE have high levels of PDE (Grewe et al., 1982; Chan et al., 1982; Sawai et al., 1995) but surprisingly some patients with AE and asthma only see an improvement in their asthma when treated with theophylline. (Giustina et al., 1984). However unpublished observations from Hanifin & Tofte suggest that other PDE inhibitors might be efficacious in AE (Hanifin & Chan 1995).

Disodium cromoglycate (DSCG) a member of the chromone family was developed to improve on the benzopyrone bronchodilator khellin which was isolated from Amni visnaga (Shapiro & Konig 1985). However DSCG is devoid of the bronochodilatory effects but prevents mast cells from releasing their mediators. This property may be due to its ability to reduce intracellular calcium in sensitized mast cells (White et al., 1984). DSCG is used in the prophylactic treatment of asthma but has been shown by one group to be effective in young children with AE when applied to the skin (Kimata & Igarashi, 1990).

ZPT is a standardized aqueous extract consisting of plant materials from ten individual herbs. Separation of this mixture by thin layer chromatography reveals that it consists of many constituents (Fig 7.1a). The individual herbs have been shown to have a variety of activities; these include anti-bacterial, anti fungal , PAF inhibition and non steroid anti -inflammatory actions (Chang & Butt 1987). Particular compounds in this mixture have had a long history of research, this includes glycyrrhetinic acid found in Glycyrrhiza uralensis which is a potent inhibitor of U p hydroxysteroid dehydrogenase (Kumagai et al .,1966). This enzyme is responsible for the conversion of cortisol to cortisone. Recently, glycyrrhetinic acid has been found to have an effect on the immune system by enhancing the IFN- y production from human lymphocytes (Shinda et al., 1986) and IL-2 production by mouse spleen cells (Zhang et al.,1993; Zhang et al.,1995). Another compound found in ZPT, paeoniflorin (P.lactiflorin) has been shown to inhibit binding of steroids to their receptors and may positively or negatively influence steroid levels through these receptors ( Tamaya et al., 1986).

4 7^ ^ ' 1' 6; C M ti\ / : /

Figure 7.1a Thin layer chromatography of ZPT. Reproduced from records from Dr W Wang

Phytopharm, Brough UK

Many acidic polysaccharides have also been isolated from the individual herbs in ZPT. These polysaccharides consist mainly of four monosaccharides L-arbinose, D-galactose, L-rhamnose and D-galacturonic acid, varying in the content of each one. Two acidic and one neutral polysacharide have been isolated from Paeonia lactiflora (Tomoda et al., 1993; Tomoda et al., 1994). Glycyrrhizans UA and UB, saposhnikovan A and C, rehmannan SA and SB have all been isolated from Glycyrrhiza uralensis , Ledebouriella seseloides and Rehmanniae ghitinosa respectively (Tomoda et al.,

1990 ;Shimizu et al., 1989a;Shimizu et al., 1989b;Tomoda et al., 1994). AU these polysaccharides and glycosides increase carbon clearance in mice and it has been suggested that they have potentiating activity on the reticuloendothelial system. Along with the aforementioned compounds, alkaloids, glycosides and tannins have been isolated from the ten herbs of which the mechanisms of actions have not yet been defined (Huang 1993).

AE patients treated with ZPT showed no change in renal or hepatic function tests after long term treatment (Sheehan et al., 1994) but there are still fears concerning the toxicity of Chinese herbs (Chan et al., 1993). Thus there is a need to isolate the active component(s). Due the role of CD23 in AE it was decided to isolate the component(s) which inhibited CD23 expression.

7.2 RESULTS

7.2.1 The effect of Glycyrrhetinic acid and paeoniflorin on