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From the Pharmacology Unit, School of Dentistry and the Pathology and Pharmacology Department, School of Medicine, Buenos Aires University; and National Research Council (CONICET), Buenos Aires, Argentina.
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Abstract |
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METHODS. Enzyme-linked immunosorbent assay (ELISA) using both rat exorbital lacrimal gland acinar cell membranes and synthetic 25-mer peptide as antigens was used to determine autoantibodies against acinar cells and M3 mAChRs. Also, nitric oxide synthase (NOS) activity was assessed to determine the biological effect of these autoantibodies in relation to the M3 mAChR.
RESULTS. Sera from dry eye primary SS (pSS) or secondary SS (sSS) patients tested by ELISA recognized membrane lacrimal gland acinar cells antigens and the synthetic 25-mer peptide, corresponding to the second extracellular loop of human M3 mAChRs. Moreover, the IgG fraction and the corresponding affinity-purified anti-M3 peptide autoantibodies from the same patients were able to activate NOS coupled to lacrimal gland M3 mAChRs. As controls, IgG and sera from women without dry eye with or without rheumatoid arthritis and from normal control subjects gave negative results on ELISA and biological assay; thus demonstrating the specificity of the reaction.
CONCLUSIONS. Autoantibodies against mAChR may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes and could be a new marker to differentiate SS dry eyes from non-SS dry eyes.
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Introduction |
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The cardinal clinical manifestations are keratoconjunctivitis sicca and
xerostomia. Keratoconjunctivitis sicca, the ocular feature of the
disease, is the result of destruction of acinar cells by periductal
mononuclear cell infiltrate.7
The lymphoproliferation
consists predominantly of CD4 T lymphocytes that transcribe IL-2,
IFN-
, and B cells that use a particular light
chain.9
10
The immune-mediated destruction of the
lacrimal glands results in severe aqueous tear deficiency that leads to
ocular surface disease and marked reduction in mucin
production.9
Ocular symptoms and signs (disabling eye irritation, foreign body sensation, burning, itching, redness, photophobia, intermittent blurring of vision) and ocular surface evaluation in SS patients are identical with those observed in non-SS dry eye patients. Moreover, the clinical course has similarities,11 but the age spread of SS is broader than that of non-SS dry eye. Also, the preponderance of female patients is much greater in SS than in non-SS dry eye.12
The criteria for the diagnosis of SS continues to be controversial.8 In the past, diagnosis has depended on the presence of typical clinical features and/or parotid gland swelling together with focal lymphocytic infiltration demonstrated on biopsy of minor salivary glands and lips. However, serologic findings recently have been recognized to have diagnostic values. Serologic findings include autoantibodies against SS-A/Ro and SS-B/La,13 antinuclear antibodies, anti–salivary gland antibodies, and rheumatoid factor.14 However, these autoantibodies have been associated not only with SS, but also with SLE, subacute cutaneous LE, congenital heart block, and neonatal lupus dermatitis.15
We have recently proposed a pathophysiological role for circulating antimuscarinic acetylcholine receptor (mAChR) autoantibodies in patients with pSS. These autoantibodies recognized and activated mAChRs in both salivary and lacrimal glands.16 17 18 There is a strong regulatory action of parasympathetic stimulation on the secretion of lacrimal and salivary glands.15 16 17 Lacrimal and salivary gland mAChRs are coupled to various signaling pathways, and the production of nitric oxide is of particular interest, because it is involved in several pathologic processes, including SS, where increased levels of nitrites are found in the saliva.19 We therefore considered it to be of interest to see whether these autoantibodies were able to stimulate nitric oxide synthase (NOS) activity in the lacrimal gland through the activation of M3 mAChRs. Furthermore, important changes in the stimulus/secretion process associated with parasympathetic stimulation have been described in an animal model of SS that could account for the reduced tear output seen in these animals.20
The aim of this work was to study the molecular interaction between serum autoantibodies from SS patients and human M3 mAChR, showing that the second extracellular loop of this receptor, is the main target of human SS autoantibody-mediated biological effects. Our results show that the presence of circulating antibodies directed to the second extracellular loop of human M3 mAChRs and that these affinity-purified anti-M3 peptide autoantibodies induce NOS activation. Also, we analyzed the distribution of anti-M3 mAChR autoantibodies in both pSS and sSS dry eye patients compared with non-SS dry eye patients, to evaluate the relationship between the presence of such circulating autoantibodies and the evidence of dry eye in SS and non-SS patients.
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Methods |
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Ophthamologic and Serologic Tests
The presence of anti-Ro/SS-A and anti-La/SS-B antibodies was a
mandatory condition to belong to group I. The diagnosis of SS followed
four or more criteria of Vitali et al.21
The following
ophthamologic tests were evaluated: Schirmer test; tear break-up time
(BUT), rose bengal staining score, and bulbar impression cytology.
Serologic tests were also performed: anti-Ro/SS-A and anti-La/SS-B
antibodies, rheumatoid factor (RF), and antinuclear antibodies (ANA).
Anti-Ro/SS-A and anti-La/SS-B were studied by double diffusion using
human spleen extract and rabbit calf thymus extract, respectively. Both
antibodies were also investigated by enzyme-linked immunsorbent assay
(ELISA) using bovine purified antigens. RF was investigated by latex
fixation and rose bengal test; ANA were investigated using an
immunofluorescence test over cryostat sections of rat liver and mouse
kidney. All the studies involving human subjects were conducted
according to the Helsinki Declaration and informed consent was obtained
from the subjects.
Preparation of Rat Lacrimal Gland Acini
Exorbital lacrimal gland acini were prepared from adult female
Wistar strain rats. Animals were used according to The Guide to the
Care and Use of Experimental Animals (DHEW Publication, NIH 80-23).
Glands were dissected away from fat, connective tissue, and lymph nodes
and immersed in a tissue chamber containing Krebs-Ringer-bicarbonate
(KRB) solution gassed with 5% CO2 in oxygen and
maintained at pH 7.4 and 30°C. All subsequent steps were performed at
4°C. Lacrimal glands were minced and incubated in KRB supplemented
with 10 mM HEPES and 5.5 mM glucose (KRB-HEPES) and 0.5% bovine serum
albumin (BSA), pH 7.4, containing collagenase (150 U/ml). Lacrimal
gland lobules were subjected to gentle pipetting. The preparation then
was filtered through nylon mesh (150-µm pore size), and the acini
were pelleted with 2 minutes centrifugation at 50g. The
pellet was then washed twice by centrifugation (50g for 2
minutes) through a 4% BSA solution made in KRB-HEPES buffer. The
dispersed acini were allowed to recover for 30 minutes in 5 ml fresh
KRB-HEPES buffer containing 0.5% BSA.22
Preparation of Microsomal Fractions
Lacrimal gland acini were homogenized for 10 seconds twice in 50
mM phosphate buffer, pH 7.4, in an Ultra-Turrax (setting 5). The
homogenate was centrifuged for 10 minutes at 1000g. The
pellets were discarded, and the supernatants were centrifuged
(10,000g) at 4°C for 10 minutes and then at
40,000g for 60 minutes. The resulting pellets were
resuspended in the same buffer supplemented with 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM ethylendiamintetracetic acid
(EDTA), 5 µg/ml leupeptin, and 1 µM pepstatin A as described
previously23
and used as a membrane source for the ELISA
test. Some experiments were performed with cardiac membranes prepared
as previously described.23
Purification of Antipeptide Antibodies by Affinity
Chromatography
The IgG fraction of 10 patients from groups I and II were
independently subjected to affinity chromatography on the synthesized
peptide covalently linked to AffiGel 15 gel (Bio-Rad, Richmond, CA).
The IgG fraction was loaded on the affinity column equilibrated with
phosphate-buffered saline (PBS), and the peptide fraction was first
eluted with the same buffer. Specific anti-M3
peptide autoantibodies were then eluted with 3 M KSCN, 1 M NaCl,
followed by immediate extensive dialysis against PBS. The IgG
concentration of the anti-M3 peptide antibodies
was detected by radial immunodifussion assay.
ELISA
Fifty microliters of peptide solution (20 µg/ml) in 0.1 M
Na2CO3 buffer, pH 9.6, was
used to coat microtiter plates (Nunc, Kastrup, Denmark) at 4°C
overnight. After blocking the wells, diluted sera from patients of
groups I, II, III, IV, and V were added in triplicate and allowed to
react with the peptide for 2 hours at 37°C. After thoroughly washing
the wells with 0.05% Tween 20 in PBS, 100 µl of 1:6000 biotinylated
goat anti-human IgG antibodies (Sigma Chemical Co., St. Louis, MO) was
added and incubated for 1 hour at 37°C. Then, a 1:6000 dilution of
ExtrAvidin-alkaline phosphatase (Sigma) was allowed to react an extra
30 minutes at 37°C. After extensive washings,
p-nitrophenylphosphate (1 mg/ml) was added as substrate, and
the reaction was stopped at 30 minutes In addition, 50 µl of
exorbital lacrimal gland acinar cell membranes (50 µg/ml) in 0.1 M
Na2CO3 buffer, pH 9.6, was
used to coat microtiter plates at 4°C overnight, and the ELISA
procedure was performed as described above. In same experiments cardiac
cell membranes (50 µg/ml; lacking in M3 mAChRs)
was used as coating antigen. Finally, the plates were read at 405 nm,
and results for each sample were expressed as the mean ± SD of
triplicate values.
Determination of NOS Activity
Female Wistar rats were used throughout. NOS activity was
measured in exorbital lacrimal glands by production of
[U-14C]citrulline from
[U-14C]arginine according to the procedure
described by Bredt and Snyder24
for brain slices. Briefly,
after a 10-minute preincubation in KRB solution, tissues were
transferred to 500 ml of prewarmed KRB equilibrated with 5%
CO2 in oxygen in the presence of
[U-14C]arginine (0.5 µCi). When antagonists
were used, appropriate concentrations of drugs were added, and tissues
were incubated for 15 minutes under carbogen at 37°C before the
addition of IgG or anti-M3 mAChR peptide. Tissues
were then homogenized with an Ultraturrax in 1 ml of medium containing
20 mM HEPES, pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM dithiothreitol, 1
mM leupeptin, and 0.2 mM phenylmethylsulphonyl fluoride at 4°C. After
centrifugation at 2000g for 10 minutes at 4°C,
supernatants were applied to 2 ml columns of Dowex AG-50 WX-8 (sodium
form). [U-14C]citrulline was eluted with 3 ml
water and quantified by liquid scintillation counting. Measurement of
basal NOS activity in exorbital lacrimal glands by the above-mentioned
procedure was 95% inhibited by 0.5 mM
NG-monomethyl-L-arginine
(L-NMMA). The results (pmol · g-1
tissue wet weight) obtained from exorbital lacrimal gland were
expressed as the difference between values in the absence or in the
presence of L-NMMA.
Drugs
A 25-mer peptide
(K-R-T-V-P-D-N-Q-C-F-I-Q-F-L-S-N-P-A-V-T-F-G-T-A-I) corresponding to
the sequence of the second extracellular loop of the human
M3 mAChR and a 24-mer peptide
(V-R-T-V-E-D-G-E-C-Y-I-Q-F-F-S-N-A-A-V-T-F-G-T-A) corresponding to the
sequence of the second extracellular loop of the human
M2 mAChR were synthesized by the PeptidoGenetic
Research Company (Livermore, CA). The synthetic peptides were
synthesized by the F-moc amino acid that was activated using
HOBt/DCO (L-hydroxy benzo
triazole/decyclohexylcarbodiimide) strategy with an automatic
peptide synthesizer (model 413A; Applied Biosystems, Foster City, CA).
The peptides were desalted, purified by high-performance liquid
chromatography, and subjected to amino-terminal sequence analysis by
automatic Edman degradation with an Applied Biosystems (model 470)
sequencer. L-NMMA was from Sigma Chemical Co., and
4-di-phenylacetoxy-N-methyl piperidine methiodide (4-DAMP)
was provided by Boehringer Ingelheim Pharmaceuticals (Berlin, Germany).
Stock solutions were freshly prepared in the corresponding buffer.
Statistical Analysis
ELISA optical density values from anti-M3
peptide antibodies were distributed in five groups. Prevalence values
between groups were compared by 
2 test. All
statistical significance was justified at P < 0.05.
Student’s t-test for unpaired values was used to determine
the level of significance. Different between means were considered
significant if P < 0.05.
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Results |
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Discussion |
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The pathogenesis of ocular surface alterations differs between the two forms of the disease. Although SS is an autoimmune disorder characterized by lymphocytic infiltration and destruction of exocrine glands,28 in non-SS dry eye, lacrimal gland biopsy shows normal morphology and no lymphocytic infiltration.29 30 Also, the gland produces tears in response to strong stimuli.27
In this regard and in this article, the differences in serologic findings including the high frequency of anti-Ro(SS-A), anti-La(SS-B), ANA, and RF, which had been recognized in patients from group I but not in those from group III, confirm the autoimmune mechanism implicated in the pathophysiology of pSS dry eyes. But, serologic markers, in particular anti-Ro(SS-A) and anti-La(SS-B), vary greatly according to the origin of the patients’ sera.15 On the other hand, these autoantibodies are associated with other connective tissue diseases.15 The presence of anti-Ro/La was a mandatory condition for group I, which is why there was a high prevalence of anti-Ro/La in this group.
The most important feature of this article relating to the autoimmune nature of SS, is the presence of autoantibodies to lacrimal gland M3 mAChRs that, acting as an "agonist-like agent," resulted in a primary, organ-specific dysfunction. Possibly, in pSS and sSS, direct M3 mAChR antibody-mediated tissue damage might occur through nitric oxide generation and accumulation, with an adverse effect on the lacrimal glands. Immunologic generation of nitric oxide could have cytotoxic effects on the cell, through the production of free radicals.31 This could have a special pathologic role, particularly in SS, where the increased release of inflammatory mediators could induce uncommonly high levels of nitric oxide.19 Although the vasodilator role of nitric oxide in the secretory process is recognized, as well as its release in normal saliva after parasympathetic stimulation32 33 34 ; nitric oxide accumulation does not appear to guarantee normal glandular function, as can be deduced from the observation that pSS patients have higher levels of nitrites in saliva,19 but its pathologic role is still unclear.
We have already reported autoantibodies against rat salivary and lacrimal glands M3 mAChR, which trigger parasympathetic, receptor-mediated biological effects.16 17 Here we have demonstrated that they are able to recognize a synthetic peptide corresponding in amino acid sequence to the second extracellular loop of the human M3 mAChR. The distribution of the amino acid sequence between rat and human M3 synthetic peptide has a great homology (84%). Moreover, the fact that an isolated fraction from pSS or sSS IgG enriched in anti-M3 peptide antibodies could reproduce the effects of the corresponding whole immunoglobulins, strongly suggests a prominent role for anti-M3 peptide antibody for the mAChR-mediated effects of total SS IgG. In addition, the synthetic peptide selectively suppressed the biological effects of SS anti-M3 peptide autoantibody and the corresponding total IgG. This supports the view that the second extracellular loop is not only the main immunogenic region of the receptor35 but can be considered essential for the biological action of these autoantibodies.
We also demonstrated in this article an association between the existence of circulating anti-M3 peptide mAChR autoantibodies and the presence of ocular symptoms, surface alterations, and a selected number of antibodies that commonly are detected in SS, making these autoantibodies a valuable marker for dry eye associated with both pSS and sSS. Tsubota et al.36 have shown a good correlation between lacrimal function, serum interleukin-2 receptor, ANA, and RF in SS dry patients.
Although evidence for desensitization of mAChR by antibodies has been reported previously,37 it is still unclear whether antibodies that interact with the second extracellular loop of mAChR are involved in the pathogenesis of lacrimal gland dysfunction. Cholinergic M3 receptor stimulation is related to secretory function including release of proteins, electrolytes, and water.38 39
It is possible that the chronic interaction of the autoantibodies with mAChR of the lacrimal gland, behaving as a muscarinic cholinergic agonist, could lead by accumulation of nitric oxide cell dysfunction or tissue damage. It is likely that, in addition to nitric oxide release, several different mechanisms may participate, including desensitization and/or downregulation of the receptor. This process could lead to a progressive blockade of mAChR an induce dry eyes, a classical sign of pSS and sSS.
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Acknowledgements |
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Footnotes |
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Submitted for publication February 14, 2000; revised May 24 and August 3, 2000; accepted August 30, 2000.
Commercial relationships policy: P.
Corresponding author: Enri S. Borda, School of Dentistry, Pharmacology Unit, Marcelo T. de Alvear 2142, 4th Floor B, 1122 Buenos Aires, Argentina. enri{at}farmaco.odon.uba.ar
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References |
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1-Adrenergic and cholinergic agonists use separate signal transduction pathways in lacrimal gland Am J Physiol 262,G1087-G1096This article has been cited by other articles:
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), 24 postmenopausal dry eye
patients without SS (
), 14 RA patients with neither SS nor dry eye
(
), and 35 normal control subjects
(<
). Sera
titers are expressed as the reciprocal of 1:2 serum dilutions from 1:10
to 1:2560 with optical density (OD) at 405 nm. Values are means ±
SD of number of patients described above.
