Systemic autoimmune disease

Central tolerance

Immature T cells migrate from the bone marrow to the thymus where they undergo thymic education as part of their developmental program. The thymus is an encapsulated, lobular organ that lies in the anterior mediastinum behind the sternum and is histologically comprised of an outer cortex and an inner medulla.

The cortex is populated with cortical thymic epithelial cells that are ectodermal in origin, as well as some bone marrow–derived macrophages along with the immature T cells. It is in the cortex where immature T cells undergo “positive selection,” wherein only the T cells that recognize self-peptides presented by human leukocyte antigen (HLA) molecules expressed on the surface of cortical antigen presenting cells (APCs) with moderate to high avidity are selected to move forward into the medulla. The T cells that display weak interactions with the self-peptide/HLA complexes in the cortex are eliminated at this stage as they are deemed unfit for immune surveillance which requires optimal recognition of foreign peptides expressed in the context of self-HLA molecules in the peripheral tissues.

The medulla is comprised of medullary thymic epithelial cells that are endodermal in origin, bone marrow–derived macrophages and dendritic cells, as well as specialized structures called “Hassall’s corpuscles” that are sites of cellular destruction. The positively selected immature T cells that enter the medulla from the cortex then undergo “negative selection.” Negative selection serves to weed out potentially autoreactive T cells by culling immature T cells that display high avidity for self-peptide/HLA complexes expressed on the surface of medullary APC. ^{, ,} A notable exception to this rule is the thymic selection of regulatory T cells (Tregs) that go on to help enforce peripheral tolerance after their egress from the thymus. Treg development in the thymus requires high-avidity interactions with self-peptide-HLA complexes on the surface of medullary thymic epithelia cells and the presence of IL-2.

For many years a dilemma in the field of tolerance centered on how negative selection was able to ensure the elimination of T cells that might react against the multitude of tissue-specific antigens present in the body. The discovery of AIRE ( A uto I mmune RE gulator), a transcription factor that induces ectopic expression of tissue-specific antigens within the thymic medulla has helped to resolve part of this puzzle. ^, Patients who are deficient in AIRE suffer from a syndrome characterized by autoimmune polyendocrinopathy, candidiasis, and ectodermal dysplasia, thus providing proof-of-principle regarding the critical role it plays in tolerance. Recent discoveries have also highlighted the role of FezF2 that can also regulate thymic medullary expression of tissue-specific antigens independently of AIRE. Furthermore, the identification of patients with combined hypomorphic and activating mutations in the T-cell signaling protein ZAP70, who display prominent autoimmune features, also suggest that alterations in T-cell signaling thresholds may disrupt the integrity of the negative-selection process in the thymus thereby potentially predisposing the host to autoimmunity.

Finally, immunologic evidence obtained from patients who have undergone thymic transplantation, where HLA matching between donor and recipient is not routinely performed, suggest that the processes of positive and negative selection may be more nuanced than prevailing dogma might suggest.

Peripheral tolerance

Silencing autoreactive T cells in the periphery is required as a back-up mechanism to regulate autoreactive T cells that escape central tolerance. This can involve T-cell intrinsic adaptive features that recalibrate signaling cascades within T cells in the face of chronic stimulation with self-antigens. Examples of such adaptive tolerance include reduced ability to mobilize intra-cellular calcium and transcription factors downstream of T-cell receptor (TCR) engagement, and re-setting the balance between kinases and phosphatases to perturb the positive signaling machinery. Additionally, potentially autoreactive T cells can also upregulate inhibitory co-receptors such as cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death 1 (PD-1), and the T-cell immunoglobulin mucin (TIM) family which elevates their activation threshold. Indeed, polymorphisms in the CTLA-4 locus have been associated with autoimmune endocrine disorders. In addition to these T-cell intrinsic mechanisms, the presence of Tregs provides an additional layer of control. As indicated above, natural Tregs are generated in the thymus and co-express CD4, CD25, and the transcription factor, Forkhead box P3 (FoxP3). Tregs can also be induced to develop from conventional naïve CD4 T cells in the periphery in the presence of certain cytokines such as transforming growth factor β (TGFβ). The importance of Tregs in suppressing autoimmune responses is highlighted in patients who harbor mutations in the FOXP3 locus as they suffer from an X-linked disorder featuring immunodeficiency, polyendocrinopathy, and enteropathy. The mechanisms that Tregs employ to dampen responses of autoreactive T cells can include CTLA-4 mediated trans-endocytosis or trogocytosis of co-stimulatory B7 molecules from the surface of professional APCs which impedes the B7-induced co-stimulatory activation signals directed to CD28 on the surface of effector T cells. There is also evidence that Tregs can limit the availability of IL-2 to other effector T cells, as well as elaborate immunosuppressive cytokines, such as TGFβ and IL-10, that bind to their cognate receptors expressed on the surface of effector T cells to attenuate effector T-cell responses. ^, Other cellular subsets that may display regulatory potential, such as natural killer T (NKT) cells and γδ T cells, have also been reported to curb disease progression in type I diabetes (T1D), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). ^,

Central tolerance

The first phase of instruction that is imparted to immature, developing B cells to help them discriminate “self” from “foreign” occurs in the bone marrow. B-cell intrinsic defects in the genes that encode for molecules associated with the B-cell receptor (BCR) and TLR pathways such as Bruton’s tyrosine kinase (BTK), adenosine deaminase (ADA), Wiskott-Aldrich syndrome protein (WASP), myeloid differentiation primary response gene 88(MyD88), interleukin 1 receptor-associated kinase 4 (IRAK-4), transmembrane activator and CAML interactor (TACI), and activation-induced cytidine deaminase (AID) impair central tolerance. Under normal circumstances, potentially autoreactive B-cell clones experience one of three different fates: clonal deletion/death, functional silencing/anergy, and “receptor editing.” Receptor editing involves an additional round of V(D)J gene segment recombination mediated by recombinase-activating genes (RAG) to replace the potentially autoreactive BCR. Recent studies involving a subset of RA patients have revealed that newly generated, circulating, naïve B cells display defects in such secondary recombination events due to aberrant activity of ataxia-telangiectasia mutated (ATM) protein that promotes repair of double-stranded DNA (dsDNA) breaks mediated by the RAG proteins. Moreover, in contrast to healthy control donors, patients with certain autoimmune disorders display marked diversity in their immunoglobulin (Ig) heavy chain variable region gene usage indicating dysregulated B-cell development. ^, Data from other carefully executed studies that have also evaluated circulating new emigrant/transitional B cells for the expression of autoreactive and/or polyreactive antibodies suggest that central tolerance checkpoints may not function optimally in untreated patients suffering from SLE, RA, T1D, Sjögren syndrome (SjS), myasthenia gravis (MG), and neuromyelitis optica spectrum disease (NMOSD). Entry of these autoreactive naïve B cells into the circulation can contribute to an environment that is permissive to the development of autoimmunity following presentation of self-antigens to circulating, autoreactive T cells as a result of ensuing somatic hypermutation of the BCR induced by this crosstalk. Multiple lines of evidence indicate that CD19 ^hi CD27 ⁻ CD21 ⁻ /lo B cells are present at high frequencies in the blood of patients with several autoimmune diseases. The presence of this subset of B cells in the circulation of RA patients correlates with an increased prevalence of erosive joint disease, and similar elevations of this B-cell subset in SLE patients are often associated with more severe autoimmune manifestations. ^, Genome wide association studies (GWAS) have also identified the 1858T polymorphism in the PTPN22 gene as a prominent genetic risk factor for the development of RA, T1D, and SLE. ^, This polymorphism can operate in a dominant-negative fashion and is associated with disruptions in BCR and TLR signaling pathways and tolerance checkpoints. Indeed, preliminary data suggest that decreased TLR9 function in SLE-associated B cells may prevent deletion of autoreactive B cells that normally occurs following cross-linking of the BCR and TLR9 with dsDNA. ^,

Peripheral tolerance

The majority of the naïve mature B cells that enter the circulation from the bone marrow require CD4 T-cell help in order to generate isotype-switched antibodies. In the absence of such help, the activation process induced following antigen exposure is often short lived and abortive, resulting in premature death of the B cell. This property of reliance on T-cell help serves to limit autoreactive B-cell activation, as it requires the presence of both the B cell and a CD4 T-cell clone that recognize the appropriate antigenic determinants derived from the same self-antigen; the likelihood that a given autoreactive B-cell clone and its companion autoreactive CD4 T-cell clone have both escaped established tolerance checkpoints is generally low. Experimental evidence also supports the notion of clonal anergy (functional silencing) of autoreactive B cells in the face of high doses of soluble antigens administered intravenously. As previously discussed with T-cell tolerance, functional silencing can involve upregulation of B-cell associated inhibitory receptors such as CD22 and FcγRIIb (CD32b) and rebalancing of positive and negative signaling cascades in the face of chronic antigen exposure and additional adaptations such as impaired migration to follicular zones within secondary lymphoid tissues that facilitate B cell and T helper cell interactions. Finally, Tregs can also suppress autoreactive B-cell activation quite likely through the same mechanism they exert toward other professional APCs as described earlier. This notion is clearly supported by the detection of circulating, autoreactive B-cell clones in patients with conditions that impair Treg numbers and/or function, such as ADA-, CD40L-, dedicator of cytokinesis 8 (DOCK8)-, HLA class II-, and WASP deficiency. ^{36 , ,}

Noninfectious triggers

The noninfectious triggers of systemic autoimmunity are very diverse. Most systemic autoimmune diseases affect more women, frequently of reproductive age, than men. This observation draws attention to the environmental etiology, especially the role of sex hormones and X-chromosome genes in autoimmune disorders. A number of studies have investigated exogenous sex hormones, silica, silicone, solvents, smoking, pesticides, mercuric chloride, and hair dyes as putative risk factors for the development of these diseases. Exposure to these agents in the environment may alter post-translational modifications, affecting immunogenicity of self-proteins and triggering an autoimmune response. For example, citrullination is linked to smoking in RA. In this study by Klareskog and colleagues, a correlation between smoking and HLA-DR SE genes was evident for ACPA-positive RA, but not for ACPA-negative RA. The combination of a history for smoking and the presence of double copies of HLA-DR SE genes increased the risk for RA by 21-fold compared with the risk among nonsmokers carrying no SE genes. In SLE, the strongest epidemiologic evidence exists for increased risk associated with exposure to crystalline silica, cigarette smoking, use of oral contraceptives, and postmenopausal hormone replacement therapy, while there is an inverse association with alcohol use. ^, Limited research evidence points to exposure to solvents, residential and agricultural pesticides, heavy metals, and air pollution to risk of SLE too. Mechanisms linking environmental exposures and SLE include epigenetic modifications resulting from exposures, increased oxidative stress, systemic inflammation and inflammatory cytokine upregulation, and hormonal effects. In other studies, estrogen replacement therapy in postmenopausal women demonstrates an increased but relatively modest risk for SSc and Raynaud disease. Environmental endocrine modulators, in the form of pesticides, may represent another opportunity for estrogen-like effects to occur, but there is scant evidence that these agents play a role in human systemic autoimmune disease.

Infectious triggers

It has long been recognized that infections may act as triggers and persistent drivers of various autoimmune diseases. A variety of different and often interrelated mechanisms by which infections trigger and perpetuate autoimmune disease in predisposed patients have been proposed. Experimental evidence suggesting that the joint may become targeted secondarily after the ACPA immune response has been initiated at another site as a consequence of an inflammatory event triggered by a common environmental exposure (periodontal infection/inflammation and/or smoking) have been reported. ^, Furthermore, ACPA-positive sera from a subset of RA patients demonstrate cross-reactivity with in vitro citrullinated peptides from Epstein-Barr virus (EBV) and human papilloma virus (HPV). ^, Prospective studies defining the kinetics of these antibody responses during natural infection will be important in assessing their role in disease pathogenesis.

In antiphospholipid syndrome (APS), studies in animal models have demonstrated the production of antibodies to β ₂ -glycloprotein I (β ₂ GPI) in response to immunization with Haemophilus influenzae, Neisseria gonorrhoeae , and tetanus toxoid. In addition, studies using peptides from microorganisms with structural similarity to β ₂ GPI demonstrate induction of β ₂ GPI antibodies, thrombosis, fetal loss, and inflammation. ^, It remains to be determined whether molecular mimicry of viral or bacterial antigens leads to the production of pathogenic anti-phospholipid antibodies (aPL) or if epitope spreading to other autoantigens is required for pathogenicity. In ANCA-associated vasculitides, an indirect mechanism of molecular mimicry leading to typical PR3-ANCA has been proposed by Pendergraft and colleagues. In selected ANCA-positive patients, antibodies against complementary peptides (antisense peptide sequence) to PR3 (cPR3) were recognized. The cPR3 peptides, which may represent mimics of microbial peptide sequences, were found to share homologies with Staphylococcus aureus sequences.

Interactions between host-specific factors (immunologic, genetics, and microbiome) and the environment play significant roles in the pathogenesis and development of autoimmune disease. Of the systemic autoimmune diseases, significant progress has been made in understanding these interactions in SLE and RA. There is evidence that other diseases such as SSc, APS, and SjS also share these attributes. Elucidating the interplay between elements involved in the diverse mechanisms associated with autoimmunity is likely to unravel optimal tools for diagnosis, prognosis, and treatment of these diseases.

Gel-based methods

Gel-based methods are based on formation of an IC between the antigen and autoantibodies that occurs within a solid phase, usually an agarose gel. There are multiple variations of this method. Radial immunodiffusion uses a gel impregnated with the relevant antigen. A well is formed in the gel, into which the patient sample is added. The proteins from the sample will diffuse in a circle into the gel, forming a concentration gradient. Optimal IC formation occurs at the zone of equivalence between the antigen and the autoantibody. If the concentration of autoantibody is sufficient, a precipitin line will be observed; a nonspecific protein stain can also be employed to improve visualization of the precipitin. Rocket immunoelectrophoresis is a variation of radial immunodiffusion. In this method, instead of allowing circular diffusion to occur, an electric field is applied to the gel, causing the proteins to migrate in a single direction from the well. This allows for improved sensitivity as the effective concentration of the autoantibody migrating into the gel is increased.

Another variation of the gel-based methods is double-immunodiffusion. In this format, the antigen of interest and the patient’s sample are placed in two adjoining wells, both of which will diffuse into the gel in a radial pattern. As the antigen and antibody migrate toward each other, ICs will form. At the point of maximal IC formation, a precipitin line may be observed. One advantage of the double immunodiffusion technique is that a patient’s sample may be assessed for multiple autoantibodies by surrounding the patient’s sample well with multiple wells, each containing a unique antigen. Counterimmunoelectrophoresis is a variation of double immunodiffusion in which an electric current is applied such that the antibody and patient’s sample are directed to migrate toward each other, rather than to diffuse radially from the well. As with rocket immunoelectrophoresis, this raises the concentration of antigen and autoantibody that will interact, thereby improving the sensitivity of the method.

Solution phase methods

Other methods based on formation of IC are immunoprecipitation and nephelometric/turbidimetric assays. For each of these methods, the IC forms between the antigen and autoantibody in the solution phase. In an immunoprecipitation assay, an IC forms between the autoantibody and a labeled form of the antigen of interest. The IC is then precipitated out of solution by altering the ionic strength of the solution by adding a salt, such as ammonium sulfate. The precipitate is collected and washed; the amount of precipitated antigen, which is proportional to the amount of autoantibody present in the sample, is measured based on the label used. This measurement is compared to a standard curve, which allows for calculation of a semi-quantitative value. A classic example of the immunoprecipitation method is the Farr assay, in which a radiolabeled form of double-stranded DNA (dsDNA) is precipitated by the presence of anti-dsDNA antibodies.

Nephelometric and turbidimetric assays are also based on the principle of IC formation, with the formation of the IC detected by light scatter. In nephelometry, the amount of light scattered is measured at a 90° angle from the incident light; the amount of scattered light detected is directly proportional to the amount of autoantibody in the patient’s sample. In turbidimetry, the light that passes through the sample (transmittance) is measured, which is indirectly proportional to the autoantibody concentration. For each of these methods, the signal is compared to a standard curve, which allows for calculation of a numeric value that is a reflection of how much autoantibody the patient has in circulation.

Indirect immunofluorescence methods

Historically, the first type of immunoassay used routinely for detection of antigen-specific autoantibodies was the indirect immunofluorescence (IIF) assay. In this assay, the source of the antigen is an intact cell or tissue which has been adhered onto a slide. Detection of autoantibody binding to the cellular substrate is accomplished through an anti-human Ig labeled with a fluorescent molecule, often fluorescein-5-isothiocyanate (FITC). The presence of an autoantibody would be identified by fluorescence of the target cell or tissue. In some cases, the autoantibody may be associated with visualization of a specific fluorescence pattern, which is related to the localization of the antigen within the cell or tissue. For some IIFs, the fluorescence pattern is reported to the patient’s medical record, depending on diagnostic relevance and antigen association. In addition, IIFs may be reported with a titer, based on analysis of serial dilutions, which reflects the amount of autoantibody present in the sample.

Line/dot blot immunoassay

In comparison to IIF, all other immunoassays use some form of purified or recombinant antigen as the capture reagent. In the line or dot blot immunoassay, the antigen is applied as either a line or dot on a membrane. This technique is analogous to a Western blot, although, in this case, the antigen is directly applied to the membrane without transfer from an electrophoretic gel. The diluted patient sample is applied to the membrane, during which time the antigen-specific antibody would be captured. After washing, an enzyme-labeled anti-human Ig antibody is added. After a second wash step, a substrate is added which is converted to a colored product on the membrane by the enzyme label. The presence of the patient’s antibody is determined by visualization of the membrane or through use of an automated reader. Although the intensity of the band or dot is related to the amount of antibody present in the patient sample, line/dot blots are often reported qualitatively. One advantage of the line/dot blot is the ability to multiplex, by including multiple antigens on a single membrane.

Sandwich immunoassay

Sandwich immunoassays refer to assays in which the patient autoantibody is “sandwiched” between an immobilized capture antigen (purified or recombinant) and a labeled anti-human Ig antibody. There are several variations of the sandwich immunoassay. In the enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA), the antigen is adhered to the bottom of a 96-well plate. Detection of the antigen-specific autoantibody binding to the antigen is performed using an enzyme-labeled anti-human Ig antibody; the enzyme label converts a colorless substrate to a colored product, the intensity of which is measured through light absorbance at the appropriate wavelength. Another format of the sandwich immunoassay is the fluoroenzyme immunoassay (FEIA). For FEIA, the antigen may be immobilized on a number of supports, including a 96-well plate. The detection antibody is also labeled with an enzyme, which converts the substrate to a fluorescent product. In the third variation of the sandwich immunoassay, the chemiluminescent immunoassay (CLIA), antigen is immobilized on paramagnetic beads. The detection antibody is labeled with a luminescent molecule, commonly isoluminol. Under appropriate conditions, the isoluminol will undergo a chemical reaction. The chemical reaction provides energy to move electrons from the ground to excited state; when the electrons return from the excited state, light is released. In all variations of the sandwich immunoassay, the signal generated from the detection antibody (absorbance, fluorescence, luminescence) is directly proportional to the amount of autoantibody that has bound to the antigen.

Multiplex immunoassays

Another type of immunoassay routinely used for autoantibody serology is generically referred to as the multiplex immunoassay (MIA). There are various formats of MIA testing; however, the most commonly used in the clinical laboratory is the addressable laser bead immunoassay (ALBIA) platform. In this type of assay, individual bead sets, each with a unique fluorescent signal, are coupled to various antigens of interest. The bead sets are then combined into a bead mixture. Autoantibody binding to the beads is detected through use of an anti-human Ig antibody labeled with a fluorescent marker, such as FITC. Measurement of the mean fluorescent intensity (MFI) for each bead, and identification of the individual bead, is accomplished by using a fluidics and laser system analogous to a flow cytometer. Identification of the bead set allows for determination of the antigen specificity of the autoantibody, while measurement of the MFI is directly proportional to the amount of autoantibody that has bound to each bead set.

Heterogeneity and antigen selection

In contrast to most protein analytes for which testing is performed in the clinical laboratory, variation between autoantibodies, both within an individual and between individuals, is significant. Within an individual, the autoantibody response is polyclonal, with production of multiple antibodies that may have different epitope specificities and binding avidities. This would be referred to as the individual’s autoantibody repertoire. To add further complexity, an individual’s autoantibody repertoire will likely change over time, due in part to epitope spreading. Epitope spreading occurs when the immune response, which was initiated against a single antigenic epitope, expands to include recognition of additional epitopes which may be from the same or different protein molecules. Between individuals, the same heterogeneity is observed. Each individual’s autoantibody repertoire will be unique. For example, consider two patients with RA. Each patient might have an immune response which leads to production of autoantibodies that bind to citrullinated peptides. However, the autoantibody repertoire from each individual will probably recognize different citrullinated peptides, possibly originating from different protein molecules. It is also probable that there will be differences in avidity with which the various autoantibodies bind to these different citrullinated peptides. Because of this complexity, designing an assay that will detect autoantibodies from both individuals can be very challenging. In addition, an assay must be able to “pick out” the relevant autoantibody from the other Igs that constitute the individual’s complete antibody repertoire. These two challenges combine to have significant impacts on the performance characteristics of an assay, specifically related to clinical sensitivity and specificity. Overcoming these challenges requires careful selection of the capture antigen. The antigen used in the assay must be broad enough to detect relevant autoantibodies that have different epitope specificities, while not including epitopes that might detect diagnostically irrelevant antibodies. Using an epitope-restricted antigen could lead to an assay with decreased sensitivity, although using a broad mixture of antigens could result in decreased specificity. Recombinant antigens may be used, although it is critical to understand the similarities and differences of the epitopes compared to the native antigen. Native antigen may be a viable option, although impurities resulting from other co-purified proteins could affect the specificity of the assay.

Characteristics of quantitation

Most immunoassays for antigen-specific autoantibodies are reported with a numeric value. The numeric value is compared to a “cutoff,” or reference interval, a result above which would be considered “positive” for the presence of the autoantibody. This numeric value is presumed to be a reflection of the concentration of the autoantibody in the patient sample. ^, However, this relationship is more complicated. In reality, most immunoassays for autoantibodies should be viewed as “semi-quantitative” methods. While the result generated from an immunoassay is determined, in part, by the amount of autoantibody, avidity of the autoantibody/antigen interaction also affects the observed signal. This “semi-quantitative” character is also evidenced in the reported result units. Virtually all immunoassays for autoantibodies report in arbitrary units, “U” or “U/mL,” rather than mass-based units. These units are generally defined by each manufacturer for their specific assay based on their calibrator material. Because of this, the patient result is only a relative comparison to the assay-specific calibrator, rather than a mass-based quantitative value.

With all these limitations, numeric autoantibody results have some diagnostic implications. For many autoantibodies, significantly elevated values are more predictive for the presence of an autoimmune disease compared to results that might be only slightly above the cutoff. In addition, in some conditions, “strongly positive” autoantibody results are linked to more aggressive disease. However, it is important to realize that most autoantibody assays are not truly “quantitative” due to complexities of the antibody avidity, antibody repertoire, and assay-specific calibration schemes.

Standardization

Standardization refers to comparability of results between different assays. For autoantibody assays, standardization can be thought of as “qualitative” or “quantitative.” Qualitative standardization requires having positive/negative agreement between assays, while quantitative standardization requires comparability of numeric values. Lack of qualitative standardization can result from differences in antigenic epitopes or assay calibration. Differences in antigens between two assays could lead to a patient sample demonstrating a relatively high positive result on one assay and a low positive or even negative result on another. This pattern of results could suggest that the patient’s autoantibody is binding to an epitope present in the antigen used in the first assay, which is not present in the antigen used for the second assay. Assay calibration can also lead to qualitative discrepancies between assays, usually around the cutoff. In other words, if one assay has a cutoff set lower to maximize sensitivity and a second assay has a cutoff set higher to improve specificity, some patient samples may have a weak positive result on one assay and a negative result on the other.

Quantitative comparability adds even further challenges to autoantibody standardization. The only way to achieve quantitative standardization is through the use of certified reference materials (CRMs). CRMs are available for only a handful of autoantibodies, and those that are available have not been routinely used as traceable calibration materials. However, it is critical to point out that the availability and use of a CRM does not guarantee quantitative standardization. This can be attributed again to autoantibody heterogeneity. Even if two assays are calibrated with traceability to the same CRM, but are detecting different subsets of autoantibodies, the quantitative results between the two methods will not be equivalent. Standardization of autoantibody assays faces significant difficulties, some of which are inherent to the autoantibody itself. Qualitative and quantitative comparability should both be considered as goals for future assay development, with the understanding that complete standardization may not be possible. For more detailed information regarding clinical laboratory test standardization, see Chapter 7 “Standardization and Harmonization.”

Epidemiology, clinical features, and classification criteria

Primary SjS is a systemic autoimmune disease typified by focal lymphocytic infiltration of the exocrine glands causing dry eyes and dry mouth. Secondary SjS is the condition in which focal lymphocytic infiltration of the exocrine glands occurs as a late complication in patients with another rheumatic disease such as RA, SLE, or SSc.

Primary SjS occurs in 0.1 to 0.6% of the general adult population with a female:male ratio of at least 9:1 and a mean age of diagnosis at around 50 years. In addition to dry eyes and dry mouth, patients may also present with symptoms of arthralgia, fatigue, and malaise. In some patients, systemic features such as inflammatory arthritis, Raynaud phenomenon, fever, and photosensitivity can be present as well.

The 2016 American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) classification criteria for SjS are summarized in Table 94.6 . Although such criteria are aimed for research, they can also be useful in clinical practice.

Autoantibodies

In a majority (>70%) of patients with SjS, antibodies to SSA/Ro and SSB/La are found. In SjS, the presence of anti-SSA alone is less common than the presence of both anti-SSA and anti-SSB antibodies. Anti-SSB alone is uncommon, and the Sjögren’s International Collaborative Clinical Alliance (SICCA) Research Groups reported that the presence of anti-SSB, without anti-SSA antibodies, had no significant association with SjS phenotypic features, relative to seronegative participants. Therefore SSB was not included in the 2016 classification criteria.

In patients with primary SjS, hypergammaglobulinemia, low C4 levels, and rheumatoid factor (RF) can be found. Thus in a patient with RF (without ACPAs), arthritis, and dryness, a diagnosis of primary SjS should be considered.

Neonatal “lupus” syndrome

Neonatal “lupus” syndrome can occur in infants born to mothers who carry anti-Sjögren syndrome A (anti-SSA/Ro) and anti-Sjögren syndrome B (anti-SSB/La) antibodies (primary SjS patient or patient with SLE) and is caused by maternal-fetal transmission of these antibodies. The neonates have a rash resembling discoid lupus erythematosus and occasionally also other abnormalities such as hepatosplenomegaly or heart block. Anti-SSA/Ro antibodies can bind to fetal heart conduction tissue and can inhibit cardiac repolarization, resulting in atrioventricular block.