Two peptides derived from the nerve growth factor precursor are biologically active.

This report provides evidence that the proregion of the NGF precursor protein contains two novel bioactive peptides. The presence of pairs of basic amino acid (aa) residues in the NGF proregion suggests that two or three peptides other than NGF may be generated by proteolytic cleavage. Synthetic peptides of 29 aa (LIP1) and 38aa (LIP2) corresponding to the sequences -71 to -43 and -40 to -3 of the proNGF, respectively, were used in this study. ELISA specific for these two peptides revealed their presence in the rat intestine. LIP1 was localized by immunohistochemistry in endocrine cells of the intestinal epithelium, and LIP2 was immunoprecipitated from an intestinal extract. We also provide evidence for the presence of specific receptors for LIP2 in several cell lines. Scatchard analysis indicated the presence of a low affinity binding site with a Kd of approximately 10(-7) M and a high affinity binding site of 10(-9) M. Cross-linking studies revealed receptor forms of about 140 kD and 93 kD in a prostatic adenocarcinoma cell line. LIP1 and LIP2 induced rapid F-actin redistribution in PC12 cells within 2 min of incubation, which suggests a role of LIP1 and LIP2 in the process of neurite outgrowth. Furthermore, both propeptides induced rapid tyrosine phosphorylation of the Trk protein in both prostatic adenocarcinoma cells and PC12 cells, thus implicating trk in their mechanism of action. These results support our hypothesis that two peptides within the NGF precursor protein are biologically active.

Evidence that natural autoantibodies against the nerve growth factor (NGF) may be potential carriers of NGF.

Nerve growth factor (NGF) was detected by enzyme-linked immunosorbent assay using the monoclonal anti-NGF antibody 27/21 in natural NGF autoantibodies (NGF NA) purified from sera of control human subjects as well as from sera of patients with rheumatoid arthritis, autoimmune thyroiditis and to a lesser degree in patients with systemic lupus erythematosus as well as in NGF NA from the synovial fluid of patients with spondylarthropathies. Our results suggest that NGF NA may be potential carriers of NGF in the circulation.

Naturally occurring antibodies against nerve growth factor in human and rabbit sera: comparison between control and herpes simplex virus-infected patients.

Antibodies against nerve growth factor (NGF) in sera were detected by enzyme-linked immunosorbent assays (ELISA), by their isolation after passage of sera through NGF immunoadsorbent columns and by their specificity to bind and immunoprecipitate mouse NGF as well as to stain by immunohistochemical methods cellular sites of NGF synthesis. Increased levels of anti-NGF antibodies were found in sera of herpes simplex virus (HSV)-infected patients but not in HSV-inoculated rabbits. As HSV latency is known to be promoted by NGF in vitro, these results may suggest that anti-NGF antibodies modulate the cytokine function of NGF and thus might play a role in HSV infection. The biological function of circulating antibodies against NGF, in general, is now open to future investigation.

Natural autoantibodies against the nerve growth factor in autoimmune diseases.

High titers of natural autoantibodies against the nerve growth factor (NGF) were detected in the sera of patients with systemic lupus erythematosus, autoimmune thyroiditis and rheumatoid arthritis. Autoantibodies to NGF from these pathological cases displayed higher avidity for NGF and a higher polyreactivity with certain cytoskeletal proteins and with DNA as compared to those from control human subjects. The biological activity, immunoglobulin composition and physiological relevance of these autoantibodies are discussed.

Nerve growth factor (NGF) autoantibodies and NGF in the synovial fluid: implications in spondylarthropathies.

We investigated the presence of nerve growth factor (NGF) autoantibodies and NGF in the synovial fluid (SF) of patients with different forms of chronic arthritis such as spondylarthropathy (SpA), rheumatoid arthritis (RA), calcium pyrophosphate dihydrate crystal deposition disease (CPPD) and osteoarthritis (OA) and compared them to their levels in serum. NGF autoantibodies were detected by ELISA and by their capacity to immunoprecipitate NGF and to inhibit its biological activity. NGF was measured with a two-site enzyme-linked immunosorbent assay. Significantly high NGF autoantibody levels (p < 10(-4)) and high frequency of detectable NGF (p < 0.01) were observed in the SF of SpA patients and to a lesser degree in RA patients as compared to CPPD and OA patients. In the serum high frequency of detectable NGF was observed only in RA patients. These results suggest a role of NGF autoantibodies and NGF in joint inflammation especially in spondylarthropathies.

Anti-NGF autoantibodies and NGF in sera of Alzheimer patients and in normal subjects in relation to age.

It has been suggested that inflammation may be a possible cause of Alzheimer's disease (AD). Increased anti-NGF autoantibody levels and increased NGF frequency in serum have previously been associated with inflammatory responses. In this study no changes in anti-NGF autoantibody titers or in NGF frequency were detected in sera of AD patients, suggesting that they are not involved in the neuroimmunological mechanisms underlying AD. There were neither age-associated changes in NGF frequency in sera of four groups of normal subjects between 18-91 years of age. In contrast, anti-NGF autoantibodies were significantly increased in sera of the 31-45 yr age group.

Increased frequency of NGF in sera of rheumatoid arthritis and systemic lupus erythematosus patients.

Nerve growth factor (NGF) levels were measured by a two-site enzyme-linked immunosorbent assay (ELISA) in sera of patients with three autoimmune diseases, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and thyroiditis. Serum NGF levels were variable (15 pg ml(-1)-1.6 ng ml-1) but not significantly different among these groups compared with control subjects. However the frequency of detectable circulating NGF was significantly higher in RA and SLE patients but not in thyroiditis patients compared with controls. The present data provides evidence for NGF involvement in two autoimmune rheumatic diseases and suggests a possible differential role of NGF as immunomodulatory agent in systemic versus certain organ-specific autoimmune diseases.

NGF involvement in ocular inflammation: secretion by rat resident retinal cells.

Nerve growth factor (NGF) has been reported to be involved in immune and inflammatory reactions. We provide evidence that NGF may play a role in ocular immune responses. Experimental autoimmune uveoretinitis (EAU) causes ocular inflammation in susceptible (Lewis, Lew x Brown-Norway (BN) F1 hybrid) but not in resistant or poorly susceptible BN and Long Evans rat strains. We examined NGF production by two resident ocular cell types, retinal Müller glia (RMG) and retinal pigmented epithelium (RPE). Interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) alone had no effect on NGF production while combined treatment with IFN-gamma and LPS strongly stimulated NGF secretion by both RMG and RPE cells. Furthermore, NGF secretion correlated with the degree of susceptibility to EAU of the different rat strains. RMG and RPE cells isolated from susceptible rats secrete high NGF levels while cells from resistant rats secrete low NGF levels.

Different mechanisms mediate the rejection of porcine neurons and endothelial cells transplanted into the rat brain.

In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.