Correlation between ribosylation of pertussis toxin substrates and inhibition of peptidoglycan‐, muramyl dipeptide‐ and lipopolysaccharide‐induced mitogenic stimulation in B lymphocytes

Abstract

Selective inhibition by pertussis toxin (PT) of mitogenic activation of mouse B lymphocytes by bacterial mitogens (peptidoglycan and lipopolysaccharide) and muramyl dipeptide (a synthetic analog of peptidoglycan fragment) was demonstrated. Mitogenic activation of B cells by protein kinase C activators and ionomycin was insensitive to PT. Also PT did not inhibit peptidoglycan- and lipopolysaccharide-induced differentiation of B cells into Ig-secreting cells, when it was added to the cultures after the proliferative stage of the response. B lymphocyte membranes contained two major PT substrates (40 and 41 kDa). The extent of PT-mediated ADP ribosylation of these substrates correlated with the degree of PT-mediated inhibition of mitogenic stimulation of B cells. B cell stimulation by all mitogens tested was not inhibited by cholera toxin at nontoxic concentrations that are known to cause maximal increase in cAMP in B cells. Since the only known substrates for PT-mediated ADP ribosylation in mammalian cells are the alpha subunits of some G proteins, our data suggest that G proteins are present in B cell membranes and that they are involved in B cell activation induced by bacterial mitogens.

Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

“…This hypothesis was further supported by the results that LTA-stimulated p42/ p44 MAPK phosphorylation was attenuated by transfection with human dominant negative mutant of TLR2 plasmid. Moreover, LPS-stimulated cellular responses have been shown to be mediated through a G i or G q protein coupling process in several cell types (10,31). This is confirmed by our observation that the LTA-stimulated p42/p44 MAPK phosphorylation was significantly attenuated by PTX but not by CTX.…”

“…LPS has been shown to be linked to a pertussis toxin (PTX)-sensitive G protein (10). To determine whether the effect of LTA on p42/p44 MAPK activation was mediated by a receptor coupled to a PTXsensitive G protein, the cells were pretreated with 100 ng/ml of PTX for 24 h and then stimulated with 50 g/ml of LTA for 7 min.…”

“…By interacting with these plasma proteins, LPS can activate several signaling pathways, including activation of phospholipase C (PLC), increase of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) (48), PKC (49), tyrosine kinase (17), p42/p44 MAPK (16,49), stress-activated protein kinase 1/JNK (15), p38/stress-activated protein kinase 2 (25), and phosphatidylinositol 3-kinase (PI 3-kinase) activity (18). In addition, several studies have demonstrated that responses to LPS in a variety of cell types may involve a pertussis toxin-sensitive G protein (10).…”

Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages.

“…Even less is known about the early intracellular events that mediate LPS responses. Several investigators have reported that LPS activates a pertussis toxin-sensitive guanine nucleotidebinding protein (G protein) (6)(7)(8)(9). However, some LPS actions in cells are unaffected by pertussis toxin treatment (refs.…”

“…Cells (1.5 x 106 per well in six-well plates) were labeled for 18 hr in 1.5 ml of growth medium containing 0.5 ,uCi of [5,6,8,9,11,12,14,15(n)-3H]20:4 (specific activity, 60-100 Ci/ mmol; DuPont/NEN). After washing in situ with PBS and addition of fresh growth medium, the cells were pretreated for 4 hr with the indicated concentration of herbimycin A.…”