Fred Elfman

Defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis.

Juvenile periodontitis (JP) is a disease characterized by severe gingival infections. PMN from some JP patients exhibit abnormal chemotactic responsiveness when challenged with the synthetic formyl peptide, FMLP. While investigating PMN function in JP, we found a patient in whom abnormal PMN chemotactic responses to FMLP were associated with a defective population of PMN formyl peptide receptor(s) (FPR). JP PMN failed to respond chemotactically when challenged with FMLP, but exhibited normal chemotactic responses upon exposure to purified human C5a. Furthermore, JP PMN were capable of degranulating and generating superoxide anion radicals as well as normal PMN upon exposure to FMLP. Binding studies demonstrated that JP PMN had a diminution in the number of high-affinity FPR. Studies in which FPR was radiolabeled by chemical cross-linking demonstrated that JP PMN FPR exhibited the same molecular weight and N-linked glycosylation as normal PMN FPR. JP PMN FPR, however, was more resistant to papain cleavage than normal PMN FPR. Autoradiograms obtained from 2D-PAGE of normal and JP PMN FPR demonstrated decreased amounts of FPR isoforms in JP PMN.

Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin).

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition.

Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane.

Human neutrophils contain subpopulations of specific granules exhibiting different sensitivities to changes in cytosolic free calcium

We examined the role of mobilization of intracellular calcium in the ability of human neutrophils to discharge specific granule constituents upon stimulation with the synthetic chemotactic factor, N-formyl-met-leu-phe. Extracellular calcium was not required for optimal secretion of the specific granule markers lactoferrin and vitamin B12-binding protein. Depletion and chelation of intracellular calcium, as well as reconstitution experiments, however, revealed different calcium requirements for stimulated secretion of these markers. N-formyl-met-leu-phe-induced secretion of vitamin B12-binding protein required half-maximal change in intracellular calcium of greater than 20 nM, while lactoferrin requirements were approximately 140 nM. Thus, it appears that cytosolic free calcium modulates fusion of subpopulations of specific granules which with the neutrophil plasma membrane.

The Role of Type IV Collagenases in Rat Bladder Development and Obstruction

The role of type IV collagenases during rat bladder development and in response to partial bladder outlet obstruction was evaluated. Gelatinase gel zymography was performed on developing rat bladders (gestation d 16 and 19, at birth, 5, 10, 15, 20, 30, and 75 d postnatally), after partial obstruction of the bladder outlet in young adults and after separation of the epithelium from the mesenchyme in young adults. Bladder function was assessed by cystometry in obstructed animals. During development, the 72-kD type-IV collagenase [matrix metalloproteinase (MMP)-2, both latent and activated] was maximally expressed in the fetal period and decreased with age; whereas the 92-kD gelatinase(MMP-9) was not expressed in developing or adult bladders. MMP-2 was localized to the bladder mesenchyme and was undetectable in isolated epithelium. In 46 obstructed rats, there was an 8-fold increase in bladder volume and weight along with smooth muscle hypertrophy (mean smooth muscle cell diameter 7.09± 0.11 μm versus 4.65 ± 0.05 μm in normal animals, p < 0.001). Obstructed rats had increased quantities of latent and activated MMP-2 and MMP-9 compared with sham-operated and normal controls. These findings suggest that expression and activation of type IV collagenases (MMP-2 and 9) are developmentally regulated and play a role in bladder remodeling during developmental morphogenesis and after partial outlet obstruction.

Decreased prostate cancer cell migration by inhibition of the insulin-like growth factor II/Mannose-6-Phosphate receptor

The 270-kDa insulin-like growth factor II (IGF-II)/cation-independent mannose-6-phosphate receptor (MPR) is a multifunctional receptor protein. Endocytoses and intracellular transport of soluble enzymes bearing mannose6-phosphate (M-6-P) residues to lysosomes is mediated by the IGF-II/MPR. We examined human prostate cancer cells for IGF-II/MPR expression to determine whether this receptor mediates cell migration. PC3 human prostate cancer cells were studied for intracellular IGF-II/MPR by immunoblotting. PC3 cell surface IGF-II/MPR expression was assessed by flow cytometric analysis. Cell motility was quantitated by a scratch migration assay, and IGF-II/MPR blockade was achieved using M-6-P or affinity-purified rabbit anti-bovine cation-independent IGF-II/MPR immunoglobulin. IGF-II/MPR is expressed in the cytoplasm and on the surface of PC3 prostate cancer cells. The mean number of PC3 cells migrating per high powered field in medium containing polyclonal anti-IGF-II/MPR immunoglobulin or M-6-P decreased significantly (5 ± 4 cells and 34 ± 5 cells, respectively) compared with control medium containing mouse immunoglobulin G (70 ± 12 cells) or mannose-1-phosphate (67 ± 7 cells). This decreased PC3 cell migration following cell surface IGF-II/MPR blockade suggests that the IGF-II/MPR may play an important role in prostate cancer cell motility.

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils.

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.