British Journal of Rheumatology 1992;31:819-822
EFFECT OF SULPHASALAZINE ON ANTIBODY RESPONSE TO ORAL ANTIGEN BY P. SHELDON*, P. PELL* AND A. McBURNEYt 'Department of Microbiology, Medical Sciences Building, University Road, Leicester LEI 9HN; tDepartment of Chemical Pathology, Glenfield General Hospital, Leicester LE3 9QP
KEY WORDS:
Rheumatoid arthritis, Inflammatory bowel disease, Cholera toxin, Sulphapyridine.
24 h) and absence of overt toxicity in the mice. Serum levels of SP were determined by high pressure liquid chromatography (HPLC). There were four groups, with 12 mice in each group. Two groups, one of which received SASP dissolved in L-lysine in their drinking water, and the other L-lysine alone, were gavaged cholera toxin, and the others were gavaged sodium bicarbonate whilst likewise receiving either SASP or L-lysine.
THE drug sulphasalazine (SASP) is widely used in the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Its mode of action remains unknown, though it appears that whereas the metabolite 5-aminosalicylic acid (5-ASA) is effective in IBD [1] this does not hold true for RA in which the other metabolite sulphapyridine (SP) has been shown to be effective [2]. It has been argued, however, that it may be the parent molecule SASP itself that induces the disease modifying effect clinically, although SP can also do this if presented to the small intestine [3]. In vitro studies have demonstrated inhibition of mitogen induced lymphocyte transformation with SASP concentrations above those found in the bloodstream, but readily achieved within the intestine. This was not found true of its metabolites SP and 5-ASA [3]. Thus it may be that SASP has an immunomodulatory effect within the gastrointestinal tract. In order to test this theory, cholera toxin, an antigen known to evoke a strong immune response within the intestine as well as systemically, was utilized. Having established this model, the effect thereon of SASP could be studied.
Dose regimen Sulphasalazine. Drinking water contained 0.16% SASP (Sigma Chemicals) dissolved in 2.0% L-lysine hydrochloride pH 7. Control mice received the same concentration of L-lysine in the absence of SASP. Both groups were on this regimen for 7 days prior to exposure to antigen. This achieved serum SP levels comparable to those found in humans receiving SASP (vide infra). Cholera toxin. Cholera toxin (CT) (Sigma Chemicals), 10 ng dissolved in 0.2 ml 0.02 M sodium bicarbonate, was gavaged at day 0, 7,14 and 21. Control mice received the same volume of sodium bicarbonate. Venous samples. Tail bleeds were carried out under light anaesthesia, on days 0,14, 21 and 28, and in half the mice on day 7.
MATERIAL AND METHODS C57BL/6J female mice, aged 8-12 weeks, were used. Preliminary experiments had established a protocol that would induce serum antibody to orally administered cholera toxin (CT). In addition, since SASP is poorly soluble in water at neutral pH, it was dissolved in lysine and administered at neutral pH. Controls received the same amount of lysine. SASP was administered via the drinking water, preliminary experiments having ensured adequate intake (on average 3.5 ml per
ELISA ELISA plates (Nunc Maxisorp Immuno), were coated overnight at 4°C with 100 uJ cholera toxin at a concentration of 1.2 ug/ml in coating buffer (Na^COj 0.159%, NaHCO 3 0.293%, NaN3 0.02%). 100 yd samples of mouse plasma at a dilution of 1:200 in serum diluent (phosphate buffered saline 5%, bovine serum albumin 0.1%, NaN3 0.02%, Tween 20 0.05%, MgCl2-6H2O 0.02%) were applied to the coated wells
Submitted 2 July; revised version acceptd 6 November 1991. Correspondence to Dr P. Sheldon.
© 1992 British Society for Rheumatology
0263-7103/92/012819 + 04 $08.00/0 819
Downloaded from http://rheumatology.oxfordjournals.org/ at Boston University on July 30, 2015
SUMMARY Cholera toxin was orally administered to mice concurrently receiving sulphasalazine (SASP) dissolved in L-lysine, or a control substance (L-lysine alone). Circulating antibodies to cholera toxin of IgM, IgG and IgA class were determined by direct ELISA at day 7, 14, 21 and 28. Although both groups made a significant antibody response to the antigen, mice receiving SASP tended to produce lower levels. These were significant for IgA on day 21 (P=0.013), and for days 7-28 (P=0.009), and 14-28 (P=0.007). Overall, considering all antibody classes together from day 7 to 28, there was a significant effect in the SASP treated group (p=
EFFECT OF SULPHASALAZINE ON ANTIBODY RESPONSE TO ORAL ANTIGEN BY P. SHELDON*, P. PELL* AND A. McBURNEYt 'Department of Microbiology, Medical Sciences Building, University Road, Leicester LEI 9HN; tDepartment of Chemical Pathology, Glenfield General Hospital, Leicester LE3 9QP
KEY WORDS:
Rheumatoid arthritis, Inflammatory bowel disease, Cholera toxin, Sulphapyridine.
24 h) and absence of overt toxicity in the mice. Serum levels of SP were determined by high pressure liquid chromatography (HPLC). There were four groups, with 12 mice in each group. Two groups, one of which received SASP dissolved in L-lysine in their drinking water, and the other L-lysine alone, were gavaged cholera toxin, and the others were gavaged sodium bicarbonate whilst likewise receiving either SASP or L-lysine.
THE drug sulphasalazine (SASP) is widely used in the treatment of rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Its mode of action remains unknown, though it appears that whereas the metabolite 5-aminosalicylic acid (5-ASA) is effective in IBD [1] this does not hold true for RA in which the other metabolite sulphapyridine (SP) has been shown to be effective [2]. It has been argued, however, that it may be the parent molecule SASP itself that induces the disease modifying effect clinically, although SP can also do this if presented to the small intestine [3]. In vitro studies have demonstrated inhibition of mitogen induced lymphocyte transformation with SASP concentrations above those found in the bloodstream, but readily achieved within the intestine. This was not found true of its metabolites SP and 5-ASA [3]. Thus it may be that SASP has an immunomodulatory effect within the gastrointestinal tract. In order to test this theory, cholera toxin, an antigen known to evoke a strong immune response within the intestine as well as systemically, was utilized. Having established this model, the effect thereon of SASP could be studied.
Dose regimen Sulphasalazine. Drinking water contained 0.16% SASP (Sigma Chemicals) dissolved in 2.0% L-lysine hydrochloride pH 7. Control mice received the same concentration of L-lysine in the absence of SASP. Both groups were on this regimen for 7 days prior to exposure to antigen. This achieved serum SP levels comparable to those found in humans receiving SASP (vide infra). Cholera toxin. Cholera toxin (CT) (Sigma Chemicals), 10 ng dissolved in 0.2 ml 0.02 M sodium bicarbonate, was gavaged at day 0, 7,14 and 21. Control mice received the same volume of sodium bicarbonate. Venous samples. Tail bleeds were carried out under light anaesthesia, on days 0,14, 21 and 28, and in half the mice on day 7.
MATERIAL AND METHODS C57BL/6J female mice, aged 8-12 weeks, were used. Preliminary experiments had established a protocol that would induce serum antibody to orally administered cholera toxin (CT). In addition, since SASP is poorly soluble in water at neutral pH, it was dissolved in lysine and administered at neutral pH. Controls received the same amount of lysine. SASP was administered via the drinking water, preliminary experiments having ensured adequate intake (on average 3.5 ml per
ELISA ELISA plates (Nunc Maxisorp Immuno), were coated overnight at 4°C with 100 uJ cholera toxin at a concentration of 1.2 ug/ml in coating buffer (Na^COj 0.159%, NaHCO 3 0.293%, NaN3 0.02%). 100 yd samples of mouse plasma at a dilution of 1:200 in serum diluent (phosphate buffered saline 5%, bovine serum albumin 0.1%, NaN3 0.02%, Tween 20 0.05%, MgCl2-6H2O 0.02%) were applied to the coated wells
Submitted 2 July; revised version acceptd 6 November 1991. Correspondence to Dr P. Sheldon.
© 1992 British Society for Rheumatology
0263-7103/92/012819 + 04 $08.00/0 819
Downloaded from http://rheumatology.oxfordjournals.org/ at Boston University on July 30, 2015
SUMMARY Cholera toxin was orally administered to mice concurrently receiving sulphasalazine (SASP) dissolved in L-lysine, or a control substance (L-lysine alone). Circulating antibodies to cholera toxin of IgM, IgG and IgA class were determined by direct ELISA at day 7, 14, 21 and 28. Although both groups made a significant antibody response to the antigen, mice receiving SASP tended to produce lower levels. These were significant for IgA on day 21 (P=0.013), and for days 7-28 (P=0.009), and 14-28 (P=0.007). Overall, considering all antibody classes together from day 7 to 28, there was a significant effect in the SASP treated group (p=
