Definition and transfer of a serological epitope specific for peptide-empty forms of MHC class I.

Nascent class I molecules have been hypothesized to undergo a conformational change when they bind peptide based on the observation that most available antibodies only detect peptide-loaded class I. Furthermore recent evidence suggests that this peptide-facilitated conformational change induces the release of class I from association with transporter associated with antigen processing (TAP)/tapasin and other endoplasmic reticulum proteins facilitating class I assembly. To learn more about the structure of peptide-empty class I, we have studied mAb 64-3-7 that is specific for peptide-empty forms of L(d). We show here that mAb 64-3-7 detects a linear stretch of amino acids including principally residues 48Q and 50P. Furthermore, we demonstrate that the 64-3-7 epitope can be transferred to other class I molecules with limited mutagenesis. Interestingly, in the folded class I molecule residues 48 and 50 are on a loop connecting a beta strand (under the bound peptide) with the alpha(1) helix (rising above the ligand binding site). Thus it is attractive to propose that this loop is a hinge region. Importantly, the three-dimensional structure of this loop is strikingly conserved among class I molecules. Thus our findings suggest that all class I molecules undergo a similar conformational change in the loop around residues 48 and 50 when they associate with peptide.

Disparate cytochemical characteristics and production of cytokines and prostaglandin E2 by human mononuclear phagocytes from the blood, lung, and peritoneal cavity.

The cytochemical characteristics and the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and prostaglandin E2 (PGE2) by peritoneal macrophages were compared with those of blood monocytes and alveolar macrophages. The comparative percentages of mononuclear phagocytes positive for peroxidase were as follows: blood monocytes > peritoneal macrophages > alveolar macrophages. The comparative percentages of cells positive for nonspecific esterase were as follows: alveolar macrophages > peritoneal macrophages = blood monocytes. The intensity of staining for nonspecific esterase was highest in alveolar macrophages and lowest in blood monocytes. Constitutive release of TNF, IL-1 beta, and PGE2 was minimal by each cell type. Lipopolysaccharide-stimulated TNF production by alveolar macrophages was approximately five times greater than that of monocytes and 10 times greater than that of peritoneal macrophages. By contrast, lipopolysaccharide-stimulated blood monocytes produced significantly more IL-1 beta than did peritoneal or alveolar macrophages. Lipopolysaccharide-stimulated production of PGE2 by peritoneal macrophages was significantly less than that of alveolar macrophages or blood monocytes. Thus peritoneal macrophages release relatively low levels of IL-1 beta, TNF, and PGE2 in response to lipopolysaccharide. Peritoneal and alveolar macrophages differ with respect to both cytochemical characteristics and lipopolysaccharide-stimulated production of TNF and PGE2 but are similar in their limited capacity to produce IL-1 beta.

Role of dialysis modality in responses of blood monocytes and peritoneal macrophages to endotoxin stimulation.

Constitutive and endotoxin (lipopolysaccharide [LPS])-stimulated release of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and prostaglandin E2 (PGE2) by blood monocytes and peritoneal cell preparations from patients on various forms of dialysis was measured. Monocytes were obtained from healthy controls (n = 20), and from patients on peritoneal dialysis (n = 8), on hemodialysis with cellulose ester membranes (n = 9), and on hemodialysis with polysulfone membranes (n = 8). Peritoneal macrophages were recovered by lavage during laparoscopic surgery from 11 healthy controls, from dialysate in 37 patients on peritoneal dialysis, and at catheter placement for transfer to peritoneal dialysis from eight patients on hemodialysis with polysulfone membranes. Peak LPS-induced production of TNF-alpha, IL-1 beta, and IL-6 by monocytes from patients on peritoneal dialysis and cellulose ester hemodialysis was less than that by monocytes from healthy controls. In contrast, monocytes from patients treated with polysulfone hemodialysis produced amounts of cytokine not different from controls. Lipopolysaccharide-stimulated PGE2 production by monocytes was the same in both patient and control groups. Peritoneal cell preparations from patients on peritoneal dialysis showed decreased release of IL-1 beta and TNF-alpha as compared with control peritoneal cells and with their own blood monocytes. To determine whether the decreased response to LPS by peritoneal cells compared with their own blood monocytes could be attributed to lower numbers of macrophages in the peritoneal cell preparations, adherence of peritoneal cells to plastic was performed. Adherence increased the percentage of macrophages from 70% to more than 90%. In monocytes and adherence purified peritoneal macrophages from peritoneal dialysis patients, no significant difference between monocytes and adherent peritoneal macrophages could be found for TNF-alpha and PGE2. Interleukin-1 beta production by the adherent peritoneal macrophages, as by total peritoneal cells, was significantly lower than that by monocytes. Constitutive production of PGE2 and IL-6 by peritoneal cells from patients on peritoneal dialysis was significantly increased. In contrast, LPS-stimulated production of TNF-alpha, IL-1 beta, and IL-6 by blood monocytes and peritoneal cells from patients receiving hemodialysis with polysulfone membranes was comparable to that produced by monocytes and peritoneal cells obtained from healthy controls. Thus, blood monocytes and peritoneal cells from patients on peritoneal dialysis and from patients on hemodialysis with cellulose-ester membranes demonstrate a decreased cytokine response to LPS, suggesting a state similar to endotoxin tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)