RecombinantDirofilaria immitisPolyprotein That Stimulates Murine B Cells To Produce Nonspecific Polyclonal Immunoglobulin E Antibody

Abstract

Nonspecific immunoglobulin E (IgE) production is an event characteristically observed in parasitic helminth infections, but its mechanisms are still unclear. To define these mechanisms, we prepared a recombinant Dirofilaria immitis protein (rDiAg) and assessed its effect on nonspecific IgE production. rDiAg preferentially induced nonspecific IgE production, without eliciting specific IgE production, as well as a Th2-type cytokine profile (

ADirofilaria immitisPolyprotein Up-Regulates Nitric Oxide Production

“…NPAs are predominantly located in the pseudocoelomic fluid of ascarids (including the porcine intestinal roundworm Ascaris suum) or on the surface of filariae (including the canine heartworm Dirofilaria immitis) and are secreted as a major component of the excretory-secretory (ES) product (7). More recently, we have found that the D. immitis polyprotein (DiAg; a Dirofilaria NPA) can bind to human CD40 molecules and induces antigen-nonspecific polyclonal immunoglobulin E (IgE) production by highly purified human and murine B cells in the presence of interleukin-4 (6,19). To verify that DiAg signaling occurs via CD40, we examined the effect of DiAg on NO synthesis by CD40-bearing and CD40-defective macrophages.…”

“…rDiAg was prepared with Escherichia coli as described previously (19). The recombinant protein was then passed through a column of polymyxin B-immobilized beads and found to have negligible endotoxin levels (Ͻ2 pg of LPS/mg of protein) as measured by Endotoxin Test-D (Seikagaku, Tokyo, Japan) (19).…”

“…rDiAg was prepared with Escherichia coli as described previously (19). The recombinant protein was then passed through a column of polymyxin B-immobilized beads and found to have negligible endotoxin levels (Ͻ2 pg of LPS/mg of protein) as measured by Endotoxin Test-D (Seikagaku, Tokyo, Japan) (19). Elicited peritoneal exudate cells were collected by washing the mouse peritoneal cavity with ice-cold RPMI 1640 medium 5 days after intraperitoneal injection with 1 ml of sterile 3% thioglycolate (Difco Laboratories, Detroit, Mich.).…”

“…Elicited peritoneal exudate cells were collected by washing the mouse peritoneal cavity with ice-cold RPMI 1640 medium 5 days after intraperitoneal injection with 1 ml of sterile 3% thioglycolate (Difco Laboratories, Detroit, Mich.). Adherent cell fractions were detached and resuspended in the culture medium as described previously (19). By nonspecific esterase staining, Ͼ95% of the cells exhibited macrophage characteristics.…”

“…Recently, CpG oligonucleotides from bacteria have been shown to induce NO synthesis by macrophages (9). To exclude the possibility that CpG contamination was responsible for the rDiAg activity, we next prepared a recombinant control protein (rCont; N-terminal half, amino acids [aa] 1 to 67, of ABA-1 [Ascaris NPA]) by the same procedure as rDiAg (19). rCont did not induce NO production at any of the concentrations used (Fig.…”