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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-01-0055.
IMMUNOBIOLOGY
From Institut National de la Santé et de la
Recherche Médicale (INSERM) U563, Department of Oncogenesis and
Signaling in Haematopoietic Cells, Institut Claude de Préval, IFR
30, Hôpital Purpan, Faculté de Médecine Purpan,
Toulouse, France.
We previously observed the presence of anti-human
µ-opioid-receptor (anti-hMOR) autoantibodies in IgG pools prepared
from several thousand healthy blood donors. These autoantibodies
behaved agonistically because of their ability to bind to the first and third extracellular loops of the receptor. In this study, we found that
each healthy donor's serum contained anti-hMOR IgG autoantibodies with
a specific activity against both the first and the third extracellular
loops of the receptor. Because of the inability of IgG to cross the
blood-brain barrier, we investigated the effects of the expression of
anti-hMOR autoantibodies on immune cells. In analogy to studies of the
effects of morphine, we investigated the ability of antibodies to
sensitize splenocytes to Fas (CD95)-mediated apoptosis. We took
advantage of the high sequence homology between murine MOR and hMOR
extracellular loops to estimate the effect on murine splenocytes of
anti-hMOR antibodies raised by immunizing mice. Splenocytes from mice
injected with Chinese hamster ovary (CHO) cells expressing MOR were
sensitized to Fas-mediated apoptosis, whereas those from mice injected
with CHO cells or phosphate-buffered saline were not. Similar
sensitization to Fas-mediated apoptosis was observed in splenocytes
from mice undergoing passive transfer either with IgG from mice
previously immunized against CHO cells expressing MOR or with IgG
directed against the first and third extracellular loops of the
receptor. Together, our data show that anti-MOR autoantibodies are
commonly expressed in healthy humans and could participate in the
control of lymphocyte homeostasis by promoting Fas-mediated apoptosis.
(Blood. 2002;100:3261-3268) Within an integrated organization, the immune
system accommodates a variety of signals originating from the
neuroendocrine system1; the immunomodulatory potency of
hormones and neuropeptides has been described
extensively.2 In turn, cytokines produced mainly by immune
cells modify the neuronal activity, as exemplified by the effect of
interleukin 1 (IL-1) on the central nervous system.3 These
communication pathways have been highlighted by the characterization on
immune and nervous cells of common receptors for cytokines, hormones,
and neuropeptides.4,5 The interactions between the nervous
and immune systems are indicated by the ability of immune cells and
neurons to synthesize cytokines, neuropeptides, and
hormones.6,7 It was reported that lymphocytes infiltrating inflamed tissues released Under normal conditions, a large part of circulating immunoglobulins
are autoreactive. Antibody activities against soluble, membrane, and
intracellular self-antigens have been observed in healthy humans and
animals.17 Among the several physiologic roles attributed
to these "natural antibodies," only their contribution to organism
protection as the first line of defense against pathogens has been
clearly established.18,19 An immunoregulatory role for natural autoantibodies has been proposed on the basis of a description of their ability to prevent ligand-receptor
binding involved in immune processes by neutralizing ligands
or receptors.20,21
We previously showed that antibodies directed against the human MOR
(hMOR) were present in pools of normal human IgG. These anti-hMOR IgG
autoantibodies displayed a specific agonistic activity because of their
simultaneous binding on the first and third extracellular loops of the
receptor.22,23 In this study, anti-hMOR IgG autoantibodies directed toward both the first and third extracellular loops of the
receptor were found in serum samples from all healthy blood donors
examined. We took advantage of the high sequence homology between
murine MOR and hMOR extracellular loops to assess the potency of
anti-hMOR IgG to sensitize immune cells to Fas (CD95)-mediated apoptosis in mice. Immunization against the hMOR was performed by
injecting mice with recombinant cells in the absence of adjuvant. Hypodiploid DNA fluorescence was used to assess apoptosis levels. Spleen cells from mice immunized against recombinant hMOR-expressing Chinese hamster ovary (hMOR/CHO) cells were sensitized to Fas-mediated apoptosis, whereas those from mice injected with either
phosphate-buffered saline (PBS) or untransfected CHO cells were not.
Sensitization to Fas-mediated apoptosis in mice injected with
recombinant hMOR/CHO cells was associated with a reduced number of
splenocytes in those mice compared with control mice injected with CHO
cells. Furthermore, splenocytes from mice that underwent passive
transfer with either IgG from mice immunized against hMOR/CHO cells or
IgG directed against the first and third extracellular loops of the
hMOR were sensitized to Fas-mediated apoptosis. Together, our data show that, under normal conditions, anti-hMOR IgG autoantibodies are expressed in human serum and could participate in the control of
lymphocyte proliferation by promoting Fas-mediated apoptosis.
Source of human IgG
Purification of human anti-hMOR antibodies
Cytofluorometric analysis of anti-hMOR antibody activity Recombinant hMOR/CHO cells and untransfected CHO-K1 cells (106) were incubated with either IgG or F(ab')2 fragments for 45 minutes at 4°C. After washing, the cells were stained with biotin-labeled, goat anti-human IgG F(ab')2-specific antibodies (Jackson Immunoresearch Laboratories). This was followed by an additional incubation with phycoerythrin (PE)-labeled streptavidin (PharMingen, San Diego, CA).For inhibition experiments, 2 peptides, corresponding to hMOR extracellular loop 1 (NYLMGTWPFGTILCK [hMOR EL1]) and loop 3 (KALVTIPETTFQT [hMOR EL3]),25 and the encephalitogenic peptide (GSLPQKSQRSQDENPVV), corresponding to amino acid residues 70 to 86 of the guinea pig myelin basic protein,26 were used. Antibodies were incubated with 20 µg/mL peptide for 1 hour at 37°C before being added to the cells for 45 minutes at 4°C. Residual antibody binding was then examined. Cytofluorometric analysis of spleen cell subpopulations After lysis of red blood cells with ammonium chloride (150 mM), potassium carbonate (10 mM), and EDTA (ethylenediaminetetraacetic acid; 0.1 mM), spleen cells were incubated with optimal concentrations of fluorescein isothiocyanate-conjugated, PE-conjugated, or biotin-conjugated monoclonal antibody (mAb) for 30 minutes at 4°C in PBS containing 1% fetal-calf serum (FCS), 5 mM EDTA, and 0.1% sodium azide. The following mAbs were used: FITC-GK1.5 anti-CD4 (TIB 207; American Type Culture Collection [ATCC], Rockville, MD), FITC-KT1.5 anti-CD8 (PharMingen), FITC-M1/70 anti-Mac1 (TIB 128; ATCC), FITC- or biotin-34.1.2S anti-Kd (HB79; ATCC), PE-Jo2 anti-CD95 (PharMingen), PE-Ha4/8 anti-keyhole-limpet hemocyanin (KLH; PharMingen), and biotin-RA3-3A1 anti-B220 (TIB146; ATCC). Spleen cells were then washed and stained with streptavidin-CyChrome (PharMingen). Data were collected on 10 000 living cells by observing forward- and side-scatter intensity on an EPICS XL flow cytometer (Beckman Coulter, Hialeah, FL) and were subsequently analyzed by using CellQuest software (Becton Dickinson, Mountain View, CA).Immunization of mice All experiments were conducted in conformity with guiding principles for the care and use of laboratory animals. Eight-week-old female BALB/c mice were purchased from Janvier (Le Genest Saint Isle, France). The mice were injected intraperitoneally with either PBS or 15 × 106 CHO cells or the hMOR/CHO cell clone every 2 weeks. Three days after the ninth injection, blood was collected and mice were killed to obtain spleens.Anti-hMOR EL1 and anti-hMOR EL3 IgG antibodies were raised by immunizing BALB/c mice with either hMOR EL1 peptide or hMOR EL3 peptide in the presence of methylated bovine serum albumin emulsified in complete Freund adjuvant (Difco, Detroit, MI). Subsequent immunizations were performed with incomplete Freund adjuvant. Determination of anti-hMOR EL IgG antibody activity Enzyme-linked immunosorbent assay plates were coated with 10 µg/mL of either hMOR EL1 or hMOR EL3 peptide in PBS overnight at 4°C. Uncoated sites were saturated with PBS containing 1% gelatin for 90 minutes at 37°C. Plates were washed and then incubated for 60 minutes at 37°C with antibody samples. Bound antibodies were revealed by using biotin-labeled, goat anti-mouse Fc -specific antibodies
(Jackson Immunoresearch Laboratories) and peroxidase-labeled streptavidin (Amersham, Slough, United Kingdom). For inhibition experiments, IgG antibodies were incubated with serial dilutions of
peptides for 60 minutes at 37°C and residual IgG binding was measured.
IgG transfers IgG purified from normal and immunized mice was injected intravenously into naive BALB/c mice. The mice were killed 24 hours later for analysis of splenocytes.Induction of Fas (CD95)-mediated apoptosis Murine splenocytes were prepared by crushing the spleen. The splenocytes were washed and enumerated by using trypan blue dye exclusion. Red blood cell lysis was performed, and 5 × 105 spleen cells were incubated in RPMI 1640 supplemented with FCS alone or containing 5 µg/mL of either hamster anti-mouse Fas (CD95) IgG mAb (Jo2 clone; PharMingen) or hamster anti-KLH IgG mAb (Ha4/8 clone; PharMingen) for 24 hours at 37°C. To better discern a potential lymphocyte sensitization to Fas-mediated apoptosis, we used a suboptimal concentration (5 µg/mL) of agonistic anti-Fas antibodies that did not induce apoptosis of splenocytes from normal and antigen-primed mice. Splenocytes were harvested, washed, and resuspended in 500 µL hypotonic fluorochrome solution (50 µg/mL propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100) overnight at 4°C. The DNA content of nuclei was examined by analyzing red fluorescence. The percentage of apoptotic cells was signified by the extent of the subdiploid DNA peak.
Anti-hMOR IgG autoantibodies with a potential agonistic activity are commonly present in serum from healthy blood donors Serum samples from 10 healthy women (22-52-years old; mean age, 31 years) and 12 age-matched healthy men (22-53-years old; mean age, 39 years) were examined for the presence of IgG directed against the hMOR. IgG from each healthy donor was prepared from serum by protein G affinity chromatography. The whole IgG fraction from each donor and control IgG (IgG pool, anti-laminin IgG, and anti-CHO-K1 IgG) showed a similar binding activity toward hMOR/CHO cells and untransfected CHO-K1 cells, as assessed by cytofluorometry (Figure 1A). Affinity purification from total IgG was necessary for observation of anti-hMOR antibodies. IgG samples from all donors were subjected to a purification procedure that included isolation of IgG directed against the recombinant hMOR/CHO cell clone, followed by depletion of IgG that reacted with untransfected CHO-K1 cells.22 As shown in Figure 1B, serum IgG from all healthy donors (men and women) contained autoantibodies directed against the hMOR. Mainly because of samples from 2 donors, a greater, though not significantly greater, IgG binding activity to hMOR was observed in men.
The hMOR, which is a 7-transmembrane domain receptor, has 4 extracellular segments (1 NH2-terminal segment and 3 extracellular loops) accessible for IgG recognition. Thus, IgG binding
to hMOR did not indicate the structures of the receptor
recognized and consequently the functional properties of antibodies.
Because the morphinelike activity of anti-hMOR antibodies purified from a pool of IgG from healthy humans depends on recognition of the first
and third extracellular loops of the hMOR,22,23 we
assessed the presence of antibodies directed toward these 2 extracellular segments in anti-hMOR F(ab')2 fragment
preparations. The binding to hMOR/CHO cells of anti-hMOR
F(ab')2 antibodies prepared from a large pool of samples
from healthy donors was inhibited by peptides corresponding to the
putative sequences of the first and third extracellular loops of the
hMOR but not by an irrelevant size-matched peptide (Figure
2A). Similar results were obtained with
anti-hMOR F(ab')2 fragments prepared from 2 randomly chosen
samples from individual donors (one man and one woman; Figure 2B and
2C). In contrast, none of the peptides neutralized the binding of
affinity-purified anti-CHO F(ab')2 fragments to recombinant
hMOR/CHO cells (Figure 2D).
We then assessed the anti-hMOR antibody specificity in IgG samples from
7 women and 7 men. As shown in Figure 3,
each donor's anti-hMOR IgG preparation contained antibodies directed
against the first and third extracellular loops. These loops were
previously identified as targets for activation of the
receptor.22 Together, these data show that anti-hMOR
antibodies with a potential agonistic activity are commonly present in
serum from healthy blood donors.
Anti-hMOR IgG sensitizes splenocytes to Fas (CD95)-mediated apoptosis Previous studies demonstrated that both exogenous opioids (morphine) and endogenous opioids promoted Fas (CD95)-mediated lymphocyte apoptosis.15,16 In analogy to these studies, we investigated the ability of anti-hMOR autoantibodies to generate similar effects. For this investigation, we developed a model in which anti-hMOR antibodies were raised by immunizing mice. The proapoptotic effect of induced anti-hMOR (auto) antibodies could be studied in murine splenocytes because IgG-induced MOR activation depends on antibody binding to the first and third extracellular loops,23 which show 93% and 92% amino acid identity, respectively, between humans and mice. In addition, we previously showed that amino acids in the hMOR constituting epitopes recognized by rat agonistic antibodies are conserved in the murine receptor.27 Our immunizing protocol to produce agonistic anti-hMOR antibodies was based on induction of experimental autoimmune Graves disease in BALB/c mice by generating agonistic anti-thyrotropin-receptor antibodies.28 In vitro, Fas-mediated apoptosis was examined on splenocytes from BALB/c mice injected with either PBS, CHO cells, or hMOR/CHO cells. Splenocytes from mice injected with recombinant hMOR/CHO cells were more susceptible to anti-Fas antibody-triggered apoptosis than those from mice injected with PBS or CHO cells (P < .01 and P < .05, respectively, on one-way analysis of variance; Figure 4A and 4B).
Anti-CHO IgG antibody activity in serum from mice injected with CHO
cells and mice injected with recombinant hMOR/CHO cells was similar, as
assessed by cytofluorometry (data not shown). The number of spleen
cells was increased in mice injected with CHO cells compared with those
injected with PBS (P < .01 on Mann-Whitney U test; Figure
5A). Spleen cellularity in mice injected
with CHO cells was also significantly greater than that in mice
injected with recombinant hMOR/CHO cells, whose splenocytes
were sensitized to Fas-mediated apoptosis (P < .05 on
Mann-Whitney U test; Figure 5A). The percentage of cells negative for
H-2 Kd in the spleen was similar in mice injected with CHO
cells and those injected with hMOR/CHO cells (5% ± 3% versus
6% ± 3%), indicating that the high number of spleen cells in
CHO-injected mice did not result from infiltrating CHO cells. The
relative number of CD4 and CD8 T lymphocytes, B lymphocytes, and
monocytes was similar in each experimental group of mice (Figure 5B).
The number of thymocytes was not modified by the immunization
procedures (Figure 5C). The sensitization of splenocytes to
Fas-mediated apoptosis observed in hMOR/CHO-injected mice was not
associated with an increase in Fas antigens on the surface of CD4 or
CD8 T lymphocytes, as assessed by cytofluorometry (Figure
6).
These data suggesting that immunization of mice against the hMOR
generated agonistic opioidlike antibodies responsible for an increased
sensitivity of lymphocytes to CD95 (Fas)-mediated apoptosis were
strengthened by IgG passive-transfer experiments. Splenocytes from mice
injected intravenously with IgG purified from pooled serum from mice
immunized against hMOR/CHO that showed splenocyte sensitization to
apoptosis in vitro were more susceptible to anti-Fas antibody-mediated
apoptosis than splenocytes from mice given IgG from PBS- or
CHO-injected mice (Figure 7).
The proapoptotic effect of IgG antibodies directed against the first
and third extracellular loops of the hMOR was then investigated. BALB/c
mice were immunized against peptides corresponding to the amino acid
sequences of either the first (hMOR EL1) or third (hMOR EL3)
extracellular loop of the receptor. The IgG binding activity measured
in serum from immunized mice was specific to the peptide used as the
immunogen. Anti-hMOR EL1 IgG bound to the insoluble hMOR EL1 peptide
but not the hMOR EL3 peptide. Reciprocally, anti-hMOR EL3 IgG bound to
the hMOR EL3 peptide but not the hMOR EL1 peptide (Figure
8A,C). The ability of anti-hMOR EL1 IgG
and anti-hMOR EL3 IgG to recognize the human as well as the murine MOR
extracellular loop was demonstrated by inhibiting the specific IgG
binding with soluble peptides corresponding to human and murine
sequences. As shown in Figure 8B, the anti-hMOR EL1 antibody activity
was equally neutralized by the hMOR EL1 peptide and a substitute of the
murine MOR EL1 amino acid sequence. Peptides corresponding to the human
and murine MOR EL3 amino acid sequences inhibited the binding of
anti-hMOR EL3 IgG to hMOR EL3 to a similar level (Figure 8D). The
indication of an absence of cross-reactivity of anti-hMOR EL1 IgG
toward hMOR EL3 and of anti-hMOR EL3 IgG toward hMOR EL1 was supported
by the inability of hMOR EL3 peptide and hMOR EL1 peptide to neutralize
the binding activity of anti-hMOR EL1 IgG and anti-hMOR EL3 IgG,
respectively (Figure 8B,D).
Anti-hMOR EL1 IgG and anti-hMOR EL3 IgG preparations were then assessed
alone or together with respect to their potency in favoring lymphocyte
apoptosis in vivo. When passively transferred into naive mice,
normal IgG and anti-hMOR EL1 IgG had no effect on Fas-mediated
splenocyte apoptosis. When compared with mice injected with normal IgG
or anti-hMOR EL1 IgG, mice injected with both anti-hMOR EL1 IgG and
anti-hMOR EL3 IgG showed a significant splenocyte sensitization to
Fas-mediated apoptosis. This suggests the presence of an
additive contribution of these 2 antibody preparations with an
essential role of anti-hMOR EL3 IgG (P = .02 on
Mann-Whitney U test; Figure 9).
Together, these data show that anti-hMOR autoantibodies present in serum from healthy blood donors could contribute to sensitivity of immune cells to Fas (CD95)-mediated apoptosis.
In a previous study, the presence of anti-hMOR antibodies in all 11 IgG samples examined, including 5 IgG pools each prepared from samples from 4 unrelated healthy blood donors and 6 single IgG fractions prepared from samples from 6 donors, led us to estimate that at least 42% of individuals express anti-hMOR autoantibodies in their serum (for each pool, we had considered that only one individual of 4 showed anti-hMOR IgG).22 In this study, isolation of anti-hMOR IgG from serum from all healthy blood donors studied revealed that these antibodies represent a regular feature of the IgG autoantibody repertoire under normal conditions. In addition, antibody-binding activity toward both the first and third extracellular loops of the hMOR responsible for opioid agonist mimicry23 was found in each anti-hMOR autoantibody sample. Thus, the morphinelike antibody activity previously observed in a pool of IgG samples from healthy donors originates from an additive contribution of all donor samples included in the pool. The low levels of circulating serum IgG in antigen-free and germ-free mice compared with mice housed in conventional conditions29 indicate that environment has a role in the occurrence of IgG antibodies under physiologic situations. Thus, it could be speculated that exposure to a wide spectrum of food antigens, drugs, and microbes results in IgG antibody responses that show some (cross-) reactivity with the first and third extracellular loops of the hMOR. Alternatively, the presence of anti-hMOR antibodies could result from a secondary anti-idiotypic response against anti-opiate antibodies30 that is generated by self-opioid or opioid peptides such as casomorphins released from bovine milk casein during digestion.31 Our inability to isolate anti-MOR IgG from pooled serum samples from normal mice, whose nutritional and medicinal intakes differ from those of humans, strengthens the evidence that environmental antigens have a role in the occurrence of detectable anti-hMOR IgG in healthy blood donors. Another possibility is that although external stimuli may enhance the production of some cross-reactive natural autoantibodies, these autoantibodies occur independently of environmental stimuli.32 Accordingly, it has been considered that disease-associated autoantibodies may emerge from the natural autoantibody repertoire.33 In this context, characterization of agonistic anti-G protein-coupled receptor autoantibodies in several pathologic conditions, including Graves disease,34 preeclampsia,35 neonatal lupus congenital heart block,36 and schizophrenia,37 may presume that these kinds of autoantibodies, including anti-hMOR autoantibodies, are a common feature of the natural autoantibody repertoire. The presence of anti-hMOR IgG antibodies with a potential agonistic
activity in the serum of all randomly selected healthy donors studied
led us to examine the putative physiologic role of these antibodies by
investigating the effects of their overexpression on murine immune
status. Taking advantage of the high sequence homology between human
and murine extracellular loops of the MOR, we immunized mice against
the hMOR to raise antibodies recognizing murine host receptors. MOR
expressed on immune cell surfaces constituted the more accessible
targets for circulating IgG antibodies that are unable to cross the
blood-brain barrier. We focused our investigation on lymphocyte
apoptosis. The cell-surface receptor Fas (APO-1/CD95) is a death
receptor that plays a key role in immune homeostasis, especially by
regulating the immune response to external and internal antigens.38 Increased Fas and Fas ligand expression in
activated macrophages and lymphocytes results in self-destruction of
those cells and therefore contributes to limitation of clonal
expansion.39,40 Similar to findings for morphine, mice
immunized with the recombinant hMOR/CHO clone showed an increased
spleen cell sensitivity to Fas-mediated apoptosis. The
essential role of IgG antibodies in this process was supported by the
observation of a similar effect in naive mice injected with purified
serum IgG. The increased sensitivity to Fas-mediated apoptosis in mice
immunized with the recombinant hMOR/CHO clone was associated with a
reduced spleen cellularity compared with that in mice immunized with
untransfected CHO cells. This finding could be perceived as an
enhancement of the Fas-dependent negative feedback of the xenogeneic
anti-CHO immune response by anti-hMOR antibodies. In addition, as was
shown in transfer experiments using anti-hMOR EL1 and anti-hMOR EL3 IgG
antibodies, the proapoptotic activity of anti-hMOR IgG antibodies could
be attributed to the recognition of both the first and third extracellular loops of the receptor. Similar to agonistic
anti- The finding of a proapoptotic effect of IgG autoantibodies specifically targeting the MOR was in agreement with results of a previous study in knockout mice showing that the MOR was the main (exclusive) mediator of the immunomodulatory effects of morphine, including spleen hypotrophy.10 As was found in mice given morphine, the reduction in the number of spleen cells observed in mice injected with hMOR/CHO was not associated with a modification in the relative content in CD4 and CD8 T lymphocytes, B lymphocytes, and monocytes.10 Because IgG cannot cross the blood-brain barrier, our results suggest that at least a part of the Fas-mediated cell death induced by opioids is mediated by receptors expressed on splenocytes. No modification of lymphocyte Fas expression was observed. The apparent discrepancy with the study of Yin et al,15 which showed an up-regulation of Fas in animals given morphine, could be attributed either to the lower discriminatory ability of the cytofluorometry method we used compared with Northern blot analysis or to the absence of a direct relation between messenger RNA (mRNA) synthesis and protein expression. Alternatively, the increase in mRNA encoding for Fas receptor observed by Yin et al in whole spleen cells from mice given morphine could have been due to an enhancement of Fas expression on cells other than T lymphocytes.44 The nature of spleen cells, which could directly interact with µ-opioid agonistic ligands, is still unclear. Although morphine acts on T-cell hybridomas,15,45 MOR mRNA has been observed clearly only on macrophages.46-48 Thus, the effect of morphine on both spleen cell subpopulations requires that either MOR is expressed on all lymphocyte subsets or Fas-mediated lymphocyte apoptosis is initiated on macrophages and then extends to lymphocytes by means of release of mediators (such as nitric oxide) able to promote up-regulation of Fas,49,50 if expression of MOR is restricted to macrophages. In contrast to splenocytes, thymocytes were not affected by
immunization of mice against hMOR/CHO cells. This observation rules out
a direct mechanism involving the MOR. Our results are in agreement with
previous findings showing that adrenalectomy abolished the apoptotic
effect of morphine on thymocytes but not on
splenocytes.16,51,52 Moreover, only Together, our findings indicate that, under normal conditions or in an IgG pool for therapeutic use, anti-hMOR IgG autoantibodies could participate in immunoregulatory processes by favoring Fas-mediated control of the immune response.
Submitted January 8, 2002; accepted June 11, 2002.
Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-01-0055.
Supported by INSERM, Université P. Sabatier, Toulouse III (ASUPS UB18CR04), and grants from Association de la Recherche contre le Cancer (ARC; 5646). G.M. is a recipient of a fellowship from ARC (P99/3).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gilles Dietrich, INSERM U563, Hôpital Purpan, Pavillon Lefebvre, Place du Dr Baylac, 31059 Toulouse, France; e-mail: gilles.dietrich{at}toulouse.inserm.fr.
1. Straub RH, Westermann J, Schölmerich J, Falk W. Dialogue between the CNS and the immune system in lymphoid organs. Immunol Today. 1998;19:409-413[CrossRef][Medline] [Order article via Infotrieve].
2.
Kohm AP, Sanders VM.
Suppression of antigen-specific Th2 cell-dependent IgM and IgG1 production following norepinephrine depletion in vivo.
J Immunol.
1999;162:5299-5308
3.
Berkenbosch F, van Oers J, del Rey A, Tilders F, Besedovsky H.
Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1.
Science.
1987;238:524-526
4.
Suzuki R, Furuno T, McKay DM, et al.
Direct neurite-mast cell communication in vitro occurs via the neuropeptide substance P.
J Immunol.
1999;163:2410-2415 5. Pozo D, Delgado M, Martinez C, et al. Immunobiology of vasoactive intestinal peptide (VIP). Immunol Today. 2000;21:7-11[CrossRef][Medline] [Order article via Infotrieve].
6.
Neumann H, Schmidt H, Wilharm E, Behrens L, Wekerle H.
Interferon
7.
Marz P, Cheng J-G, Gadient RA, et al.
Sympathetic neurons can produce and respond to interleukin 6.
Proc Natl Acad Sci U S A.
1998;95:3251-3256
8.
Cabot PJ, Carter L, Gaiddon C, et al.
Immune cell-derived
9.
Shavit Y, Depaulis A, Martin FC, et al.
Involvement of brain opiate receptors in the immunosuppressive effect of morphine.
Proc Natl Acad Sci U S A.
1986;83:7114-7117
10.
Gavériaux-Ruff C, Matthes HWD, Peluso J, Kieffer BL.
Abolition of morphine-immunosuppression in mice lacking the µ-opioid receptor gene.
Proc Natl Acad Sci U S A.
1998;95:6326-6330
11.
Govitrapong P, Suttitum T, Kotchabhaki N, Uneklabh T.
Alterations of immune functions in heroin addicts and heroin withdrawal subjects.
J Pharmacol Exp Ther.
1998;286:883-889
12.
Grimm MC, Ben-Baruch A, Taub DD, et al.
Opiates transdeactivate chemokine receptors:
13.
Roy S, Cain KJ, Chappin RB, Charboneau RG, Barke RA.
Morphine modulates NF 14. Matthes HWD, Maldonado R, Simonin F, et al. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid-receptor gene. Nature. 1996;383:819-823[CrossRef][Medline] [Order article via Infotrieve]. 15. Yin D, Mufson A, Wang R, Shi Y. Fas-mediated cell death promoted by opioids. Nature. 1999;397:218[CrossRef][Medline] [Order article via Infotrieve].
16.
Yin D, Tuthill D, Mufson R, Shi Y.
Chronic restraint stress promotes lymphocyte apoptosis by modulating CD95 expression.
J Exp Med.
2000;191:1423-1428 17. Avrameas S. Natural autoantibodies: from `horror autotoxicus' to `gnothi seauton.' Immunol Today. 1991;12:154-159[Medline] [Order article via Infotrieve].
18.
Boes M, Prodeus AP, Schmidt T, Carroll MC, Chen J.
A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection.
J Exp Med.
1998;188:2381-2386
19.
Ochsenbein AF, Fehr T, Lutz C, et al.
Control of early viral and bacterial distribution and disease by natural antibodies.
Science.
1999;286:2156-2159 20. Hansen MB, Svenson M, Abell K, Yasukawa K, Diamant M, Bendtzen K. Influence of IL-6 autoantibodies on IL-6 binding to cellular receptors. Eur J Immunol. 1995;25:348-354[Medline] [Order article via Infotrieve].
21.
Svenson M, Hansen MB, Ross C, et al.
Antibody to granulocyte-macrophage colony-stimulating factor is a dominant anti-cytokine activity in human IgG preparations.
Blood.
1998;91:2054-2061 22. Macé G, Blanpied C, Emorine LJ, Druet P, Dietrich G. Isolation and characterization of natural huma | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
-endorphin, which produced analgesia by
acting on opioid receptors (7-transmembrane segment,
Gi/o-protein-coupled receptors) expressed on peripheral afferent
nerves.
), the value obtained with a dilution at which CHO-K1
cell staining was similar to the background. Controls were the specific
antibody-binding activity of unpurified IgG (
), affinity-purified
anti-laminin IgG (
), affinity-purified anti-CHO IgG (
), and
affinity-purified anti-hMOR IgG (
) prepared from a pool of
human IgG.
), CHO cells (
), or hMOR/ CHO
cells (
), anti-hMOR EL1 IgG
(
and 
opioid-receptor subclasses have been observed on thymic
cells.