Morton A. Kapusta M.D., FRCPC, FACP

Editorial:  By Dr. MORTON A. KAPUSTA LEARNING OBJECTIVE To ease the reader from the title, through a complex study, involving multiple techniques, toward an interesting hypothesis. OVERVIEW OF THE STUDY This Swedish study examined the relationship between Shared Epitope (SE), Antibodies to Citrullinated Protein/Peptides, Cigarette Smoking and Rheumatoid Arthritis (RA). The authors compared 829 patients with newly diagnosed RA, prior to the institution of disease modifying anti-rheumatic drugs, with 631 normal controls.

Smoking histories were obtained; bronchial washings were studied, sera were analyzed for antibodies against Cyclic Citrullinated Protein (CCP), and cellular DNA was analyzed for the DRB1x01, DRB1x04 and DRB1x10 alleles of the HLA-DRB1 gene.


BACKGROUND AND THE PRESENT EXPERIMENTAL STUDIES

(A)    Citrullinated Protein/Peptides (1,2,3): A number of different Peptidylarginine Deiminase (PAD)  enzymes remove a nitrogen-containing imino group from Argenine, and substitute an oxygen-containing carbonyl group:  Argenine is thus converted into Citrulline.  

Patients with RA are quite unique in their ability to produce a variety of antibodies against Citrullinated Protein/Peptides.

Although CCP does not exist in nature, commercially available kits containing this antigen are highly sensitive, and specific, for RA, in research studies.  A specificity of  98.5% will result in a sensitivity of 73.7% for RA.  The present study used a commercial ELISA assay to determine the presence of antibodies against CCP.


(B)    SMOKING:  

Smoking predisposes toward the development of RA (4).  

In the present study, bronchiolar lavage cells were obtained from smokers and non-smokers.  The presence of citrullinated protein in these cells was determined by immunohistochemical techniques.


(C)  SHARED EPITOPE (SE) (5,6,7,8)

The Major Histocompatibility Complex (MHC) is a cluster of Genes on chromosome 6, which encode the various Human Leukocyte Antigens (HLA). The term MHC serves to emphasize that much of the original work on HLA molecules, and their Genes, was done within the context of transplantation studies.

Some of the pioneer work on tissue typing used the mixed lymphocyte response.  Mixing lymphocytes from two individuals with different HLA-D antigens induces the lymphocytes to proliferate.  Mixed lymphocytes from two patients with RA will often not stimulate each other, suggesting an identity of their HLA-D surface antigen.  

Stastny, found two different HLA-D determinants in his RA patients: the most common showed HLA-D identity with 48% of patients with RA, vs 8% of controls;  the second, showed identity with a different 20% of RA patients, vs 4% of controls.  The first of these HLA-D antigens was initially termed as HLA-DW4.  Serological studies for the HLA antigens, which were done in a later era, renamed this HLA antigen as HLA-DR4.

Crystallographic studies demonstrated that HLA-D cell surface molecules are composed of alpha and beta chains.  These two chains form a pocket, which presents antigen to helper T-Lymphocytes.  The configuration of the pocket determines the shape of the antigen, which it will contain.  In turn, the amino acid sequence of beta chain is the major determinant of the structure of this pocket.  The genetic code for the beta chain is found in the HLA-DRB1 gene.  DNA sequencing of the nucleic acid composition of the various Alleles of the DRB1 gene demonstrated that their nucleic acids coded a common sequence of amino acids, for the beta chain, in patients with RA.  This common sequence of amino acids, found in RA, is termed the SE.  

The Allele, of the HLA-DRB1 Gene, responsible for the above-mentioned HLA-DR4 Antigen is termed DRBx0401 (the number 01, because it was the first one to be discovered).

The present study performed genotyping for the following SE alleles of the HLA-DRB1 gene:  DRB1x01; DRB1x04; and DRB1x10.  The authors did not type the alleles beyond this point, “for practical reasons”.  Any genotype with a combination of two of these alleles was considered a double SE Genotype.  


RESULTS

As seen in table 2, in the results, 504 of the patients with early RA (61%) had CCP antibodies; 325 (39%) did not.

Of those with CCP antibodies, the presence of one SE increased the relative risk (RR) of RA to 3.3; and two SEs to 5.4.  Smoking increased the RR associated with one SE to 6.5; and with two SEs, to 21.0.

Conversely, in the absence of CCP antibodies, neither SE, nor smoking, increased the RR of RA.  

Cells, in bronchial washings obtained from smokers, demonstrated citrullinated protein/peptides, while those from non-smokers did not.


DISCUSSION

61% of patients with early RA, in this study, had CCP antibodies.  This is in agreement with previous work, which found CCP antibodies in 58.9% of early RA patients.

That two alleles for SE carried a greater RR for RA, than one Allele, is also in agreement with previous studies.  The presence of a dose-response effect of SE Alleles supports also their role in pathogenesis of RA.

The authors offer an hypothesis, based largely upon animal experiments done on HLA-DR4-IE transgenic mice, in Dr. David Bell’s laboratory (9,10,11).

HLA-DR4-IE transgenic mice, with the DRB1x0401, Allele were studied.  They did not express mouse MHC in the antigen-binding site.  The antigenicity of citrullinated, and non citrullinated, Vimentin Peptide, was studied in these mice.  Vimentin was chosen, largely, because antibodies to its citrullinated peptide are found in RA ( Anti Sa Antibodies).  The citrullinated, but not the non-citrullinated peptide, could sensitize T-lymphocytes in these mice.  The immune response was specific for the citrullinated peptide.  Moreover, the citrullinated peptide had a 90 fold higher affinity for the SE pocket, than the non-citrullinated peptide.  

In other experiments, the same type of transgenic mice were immunized with citrullinated, or non-citrullinated, Fibrinogen.  Fibrinogen was studied, because it is found in joint inflammation.  Forty percent of the mice immunized with citrullinated Fibrinogen, but none of the non-citrullinated Fibrinogen group, developed arthritis.  

Based upon these animal experiments, and their findings in patients with early RA, the authors proposed the following working hypothesis:

“Long-term exposure to cigarette smoke, and probably also to other environmental stimuli, may induce mechanisms that accelerate deimination of Arginine to Citrulline in autoantigens present in the lungs, possibly via up-regulation of peptidylarginine deiminase activity in Macrophages that are activated or undergoing apoptosis.  An immune response to the citrullinated protein might then be preferentially induced in individuals carrying the HLA-DR SE genes, since citrullination has been demonstrated to increase the binding of modified peptides to SE – containing HLA DR antigens and thereby to enhance the immunogenicity of the protein. -----------.”

---Induction of systemic immunity to citrullinated proteins precedes the disease, and in which a second event may trigger an undifferentiated arthritis, which is accompanied by citrullinated of proteins in the synovium.  This synovitis may be transient in individuals without preceding anticitrulline immunity, while development toward the chronic disease we call RA is more likely in individuals with pre-existing citrulline immunity, which may enhance joint inflammation.”


SUMMARY

SE and cigarette smoking seem to be relevant in the pathogenesis of RA only in individuals capable of making antibodies to CCP.  This accounts for 61% of the early RA patients in this study.  The remainder of the early RA patients, with the same SE, did not have these antibodies.  Neither SE, nor smoking increased the RR of RA in patients without these antibodies.  

If the present working hypothesis is correct, then the immunopathogenesis of RA is different in patients with antibodies to Citrullinated Protein/Peptides than from those without these antibodies.

ADDENDUM - May 5, 2006

1. Meyer O, Labarre C, Dougados M, et al.  Anticitrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage.  Ann Rheum Dis 2003; 62:  120-126.
2. DeRycke L, Peene T, Hoffman IEA, et al.  Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis:  diagnostic value, associations with radiological progression rate, and extra-articular manifestations.  Ann Rheum Dis 2004; 63:  1587-1593.
3. Braun-Moscovici Y, Markovits D, Zinder O, et al.  Anti-Cyclic Citrullinated Protein Antibodies as a Predictor of Response to Anti-Tumor Necrosis Factor-ά Therapy in Patients with Rheumatoid Arthritis.  J Rheumatol 2006; 33:  497-500.
4. Wolfe F.  The Effect of Smoking on Clinical, Laboratory, and Radiological Status in Rheumatoid Arthritis.  J Rheumatol 2000; 27:  630-637.
5. Stastny P.  Mixed Lymphocyte Cultures in Rheumatoid Arthritis.  JCI 1976; 57: 1148-1157.
6. Winchester R.  Genetic Determination of Susceptibility and Severity in Rheumatoid\ Arthritis.  Ann Int Med 1992; 117:  864-870 [editorial].< style="font-family: arial;">
7. Harris ED.  Excitement – and Confusion – about HLA and Rheumatoid Arthritis.  Ann Int Med 1995; 232-233 [editorial].
8. Gourraud P-A, Boyer J-F, Barnetche T, et al.  A New Classification of HLA-DRB1 Alleles Differentiates Predisposing and Protective Alleles for Rheumatoid Arthritis Structural Severity.  Arthritis Rheum 2006; 54:  593-599.
9. Hill JA, Southwood S, Sette A, et al.  Cutting Edge:  The Conversion of Arginine to Citrulline Allows for a High-Affinity Peptide Interaction with the Rheumatoid Arthritis – Associated HLA-DRB1x0401 MHC Class II Molecule.  J Immunol 2003;178:  538-541.
10. Hill JA, Wehrli B, Jevnikar AM, et al.  Citrullinated Fibrinogen Induces Arthritis in HLA DRBx0401 Transgenic Mice.  Arthritis Rheum 2003; 48:  Suppl 9:  S348 #832.
11. Bell DA.  Personal Communication.  Mar. 2006.

A new model for an etiology of rheumatoid arthritis: Smoking may trigger HLA-DR (shared epitope)-restricted immune reactions to autoantigens modified by citrullination
Lars Klareskog, MD, PhD 1 *, Patrik Stolt, MD, PhD 2, Karin Lundberg, PhD 1, Henrik Källberg, MSc 3, Camilla Bengtsson, MSc 3, Johan Grunewald, MD, PhD 1, Johan Rönnelid, MD, PhD 4, Helena Erlandsson Harris, MD 1, Ann-Kristin Ulfgren, PhD 1, Solbritt Rantapää-Dahlqvist, MD, PhD 5, Anders Eklund, MD, PhD 1, Leonid Padyukov, MD, PhD 1, Lars Alfredsson, PhD 3, the Epidemiological Investigation of Rheumatoid Arthritis Study Group

Abstract

Objective
To investigate whether smoking and HLA-DR shared epitope (SE) genes may interact in triggering immune reactions to citrulline-modified proteins.

Methods
In a case-control study involving patients with recent-onset rheumatoid arthritis (RA), we studied interactions between a major environmental risk factor (smoking), major susceptibility genes included in the SE of HLA-DR, and the presence of the most specific autoimmunity known for RA (i.e., antibodies to proteins modified by citrullination). Immunostaining for citrullinated proteins in cells from bronchoalveolar lavage fluid was used to investigate whether smoking is associated with citrullination in the lungs.

Results
Previous smoking was dose-dependently associated with occurrence of anticitrulline antibodies in RA patients. The presence of SE genes was a risk factor only for anticitrulline-positive RA, and not for anticitrulline-negative RA. A major gene-environment interaction between smoking and HLA-DR SE genes was evident for anticitrulline-positive RA, but not for anticitrulline-negative RA, and the combination of smoking history and the presence of double copies of HLA-DR SE genes increased the risk for RA 21-fold compared with the risk among nonsmokers carrying no SE genes. Positive immunostaining for citrullinated proteins was recorded in bronchoalveolar lavage cells from smokers but not in those from nonsmokers.

Conclusion
We identified an environmental factor, smoking, that in the context of HLA-DR SE genes may trigger RA-specific immune reactions to citrullinated proteins. These data thus suggest an etiology involving a specific genotype, an environmental provocation, and the induction of specific autoimmunity, all restricted to a distinct subset of RA.
Received: 23 March 2005; Accepted: 20 June 2005