Efeito secretório intestinal do sobrenadante de macrófagos estimulados com a toxina A do clostridium difficile: papel da interleucina-1B

Abstract

The Clostridium difficile produces two exotoxins denominated toxin A (TxA; 308 kDa) and toxin B (TxB; 279 kDa). The mode of intestinal action of those toxins is not still totally understood, nevertheless several scientific studies have confirmed the involvement of the same ones, in the pathogenesis of the diarrheal diseases. In the beginning of this work it was determined the intestinal secretory effect induced by the supematant of macrophages stimulated with TxA or TxB, in rabbit ileum mounted in Üssing chambers. From this protocol it was observed that the supematant of macrophages stimulated with TxA (3,2xlO'7M, 9,6x10'7M and 3,2x10"6M) cause a potent intestinal secretion (AIsc = 41,0, 52,0 and 90,0 pA.cm'2, respectively). However, the supematant of macrophages treated with TxB (3,6xl0‘ M) did not alter significantly this activity (AIsc = 28,0 pA.cm' vs. AIscCOntroi= 20,0 pA.cm'2). It is worth pointing out that the addition of toxin TxA (3,2x10_<’M) directly in the Üssing chambers did not produce intestinal secretion (AIsc= 2,2 pA.cm'2). In addition, the genesis of the factor of intestinal secretion (FSI), present in this supematant, was blocked (80%) by the incubation of TxA with the PCG4. In the following stage it was investigated the involvement of G protein in the genesis of FSI, through a previous treatment of the macrophages with the active pertussis toxin. So, it was evidenced that this procedure is able to block the release of FSI (blockade: 61%). Following, it was evaluated the effect of several pharmacologics blockers on the synthesis of FSI. Thus, it was observed that specific inhibitors, for example, inhibitor of protein synthesis (reduction: 67%), proteases (57%), phospholipase A2 (54%), cyclooxygenase (62%), cyclo and lipoxygenase (48%), TNF-a synthesis (48%) and PAF antagonist (55%), reduce the synthesis and release of FSI. In addition, it was evidenced that, such as IL-lra (80%), the monoclonal iv antibody anti-IL-lp (72%), but not anti-EL-la (p> 0,05), blocked signifícantly the secretory effect of FSI. In the subsequent stage it was tried to identify the FSI, through ELISA method. In that way, it was observed that TxA (3,2x10'7 M, 9,6x10' 7M and 3,2x10'6M), but not TxB (3,6xlO'7M), is capable to stimulate the synthesis of IL-ip (665,0, 413,0 e 3477,0 pg/ml, respectively). It was also evidenced, that the supematant of macrophages stimulated with TxA (3,2xl0‘7 M and 3,2x107’ M) or TxB (3,6x10'7M) presents high leveis of TNF-a (803,0, 1040,0 and 555,0 pg/ml, respectively). At last, it was dertermined the mechanism of action of FSI, through the previous treatment of the ileum mucosal with indomethacin, bumetanide or tetrodotoxin. Thus it was observed that the activity of FSI is dependent on the Cl' transport; and that this effect could be blocked with indomethacin (88%), as well as with tetrodotoxin (84%). Therefore, the data of this work demonstrate that macrophages stimulated with TxA synthesizes IL-lp which is able to provoke intestinal secretion. They, also, demonstrate that the genesis of this cytokine is dependent of the activation of a G protein; and that PAF, prostaglandins and TNF-a are involved in this event. In addition, this secretory activity is dependent on the prostaglandins synthesis, by resident cells, and activation of the enteric nervous system.