Identification of mu-opioid receptor epitopes recognized by agonistic IgG.

We have previously reported the presence of IgG antibodies with a morphine-like activity in the serum of healthy individuals. The agonistic activity of IgG was dependent on their binding to the first and the third extracellular loops of the human mu opioid receptor. In this study we show that IgG antibodies obtained by immunizing rats with peptides corresponding to these two loops exhibited a similar morphine-like activity. Residues corresponding to Y(130), M(132), G(133), T(134) within the first and F(315) within the third extracellular segment were required for antibody binding and conferred to IgG a high mu-opioid selectivity.

Morphine-like activity of natural human IgG autoantibodies is because of binding to the first and third extracellular loops of the mu-opioid receptor.

We have previously demonstrated that randomly selected healthy individuals express anti-human mu-opioid receptor antibodies which behave as agonist in vitro. In this study, we show that the activity of these antibodies was not affected by the deletion of the amino-terminal region of the receptor. Using agarose-bound peptide columns, we affinity-purified IgG specifically directed toward each extracellular loop. Whatever its specificity, each anti-human mu-opioid receptor (hMOR) extracellular loop peptide IgG preparation was unable, when examined individually, to reduce adenylate cyclase activity. Activation of the hMOR was, however, achieved by the simultaneous binding of IgG to the first and third extracellular loops of the receptor. Our results suggest that the simultaneous binding of IgG antibodies to these two loops mimics morphine-induced receptor activation by triggering a coordinated shift of the third and sixth transmembrane helices.

Isolation and characterization of natural human IgG with a morphine-like activity.

Although naturally occurring antibodies have been associated with numerous biological activities, their functional relevance is still a matter of debate. The presence of natural autoantibodies towards immune-related molecules such as cytokines and antibodies suggests a physiological immunomodulatory role. The neuroendocrine opioid system participates in the immune homeostasis. We report here the presence of antibodies with an agonist-like activity towards the human mu-type opioid receptor within a normal human IgG pool. Starting from an IgG pool, autoantibodies were affinity purified using Chinese hamster ovary cells expressing the human mu-opioid receptor. Their specificity was assessed by cytofluorometry and pharmacological analyses. The potency of these antibodies to recognize the mu-opioid receptor was similar to mu-opioid selective agonists. Furthermore, the functional opioid-like activity of the anti-opioid receptor IgG was demonstrated by their ability to inhibit adenylate cyclase activity by a Gi/o-protein-mediated mechanism as indicated by abrogation of the effect by either opioid antagonist or pertussis toxin. Five IgG pools, each from four unrelated healthy blood donors, and single IgG preparations from six other donors were prepared. Antibodies directed against the mu-opioid receptor were found in all IgG samples.

Antifection: an antibody-mediated method to introduce genes into lymphoid cells in vitro and in vivo.

We have developed a simple, safe and versatile method, termed antifection, by which antibodies are used as delivery vehicles to introduce genes into cells expressing specific surface antigens. Antibodies directed against CD3, CD34 or surface immunoglobulins were covalently coupled to plasmids containing marker genes (neoR, beta-galactosidase). Such conjugates were used in vitro and/or in vivo to antifect (transfect using antifection) cells bearing the respective targeted epitope on either normal splenic B lymphocytes or lymphoid-related cell lines. In these conditions the expression of the protein encoded by the marker gene was readily detected. Antifection is a method of delivering genes through a physiological cellular pathway, receptor-mediated endocytosis, into specific cell types, and thus may be considered as an alternative for gene therapy strategy.

Serum IgE concentration and other immune manifestations of treatment with gold salts are linked to the MHC and IL4 regions in the rat.

A subset of patients with rheumatoid arthritis occasionally develops skin reactions and glomerulonephritis and exhibits an increase in serum IgE concentration when treated with gold salts. Brown-Norway (BN) rats injected with aurothiopropanolsulfonate (ATPS) also manifest an autoimmune glomerulonephritis and increased serum IgE concentration, whereas Lewis (LEW) rats are resistant to complications. Here, we show linkage between responses to ATPS in a (BN x LEW) F2 cohort and the major histocompatibility complex (RT1) on rat chromosome 20 and between markers in the region of IL4 and other candidate genes on rat chromosome 10. Recently, human serum IgE concentration has been reported to be linked to the IL-4 region. Taken together, these findings raise the possibility that homologous genes could be implicated in ATPS manifestations in the rat and in the regulation of IgE levels in the human.

Genetic study of gold-salt-induced immune disorders in the rat.

BACKGROUND: Rheumatoid arthritis patients treated with gold salts occasionally develop a glomerulonephritis and an increase in serum IgE concentration. Brown-Norway (BN) rats injected with aurothiopropanolsulphonate (ATPS) exhibit an increase in serum IgE concentration, produce antilaminin antibodies (Abs) and develop glomerular linear immunoglobulin (Ig) deposits, occasionally a membranous glomerulopathy and vascular granular Ig deposits. Lewis (LEW) rats are resistant. METHODS: The genetic requirements governing the appearance of these manifestations were studied in congenic rats, and in F1 hybrids injected with ATPS. RESULTS: Non-MHC-linked genes from the BN strain were absolutely required for all the traits to be observed. The RT1n (BN) or RT1(1) (LEW) haplotypes at the MHC were permissive for all the manifestations to appear and two RT1(1) alleles were associated with the highest response. However, granular Ig deposits were only observed in RT1n rats. The high serum IgE concentration and the antilaminin Ab level were associated with the presence of glomerular Ig deposits but were not associated with the presence of vascular Ig deposits. CONCLUSIONS: This study shows that susceptibility to ATPS was mainly dependent upon non-MHC-linked BN genes and that the involvement of MHC-linked genes differed depending upon the character considered. There is an epistatic effect between the various genes.

Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. II. Opposite effect of interleukin-2 and interleukin-4.

In susceptible animals evidence is accumulating for a primary role for Th2 cells in the course of HgCl2-induced autoimmunity, and for a contribution of Th1 cells in the self-regulated phase of this disease. We have reported that incubation of 2B4.11 T cell hybridoma with HgCl2 induced programmed cell death. This paper shows that recombinant IL-2 significantly diminished HgCl2-induced 2B4.11 cell death. Although no effect was observed upon incubation with exogenous IL-4, we observed a significant protection by adding an anti-IL-4 monoclonal antibody to the culture. Accordingly, by RT-PCR we found the presence of IL-2 receptor-encoding mRNA, and by cytofluorometry, the expression of the protein was detected only after exposure to HgCl2. Moreover, upon HgCl2 treatment, 2B4.11 cells were induced to produce IL-4. Altogether these findings showed that cytokine environment, IL-2, IL-4 otherwise defining the Th1/Th2 dichotomy, in conjunction with a chemical may differentially influence the fate of cell populations, death or survival.

Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. I. Effect of the proto-oncogene Bcl-2.

Mercuric chloride (HgCl2) as well as several drugs can induce T cell activation leading to systemic immune-mediated diseases in genetically susceptible individuals or rodents. T cell hybridomas represent a well-characterized model system for in vivo mechanisms of various stimuli-induced cell death. The cellular response to HgCl2 was examined using various T cell lines and particularly the murine T cell hybridoma 2B4.11. Exposure to HgCl2 induced both necrosis and apoptosis in a dose- and time-dependent way as demonstrated by DNA fragmentation analysis, flow cytometry of the whole cells and of isolated nuclei, and morphological examination. HgCl2-induced cell death was partly inhibited by cycloheximide. The expression of human Bcl-2 in 2B4.11 cells after transfection significantly prevented HgCl2-induced cell death but did not affect the susceptibility to apoptosis induced by an anti-CD3 epsilon mAb. Subcytotoxic doses of HgCl2 enhanced metabolic activity of Bcl-2 transfectants in contrast with mock-transfected cell line. Thus, we conclude that apoptosis is part of the cell death process induced by HgCl2 and that the ability of Bcl-2 to prevent the death of one particular cell line is stimulus-dependent suggesting the existence of different pathways leading to cell death.