Independent association of anti–β2-glycoprotein I antibodies with macrovascular disease and mortality in scleroderma patients

Authors


Abstract

Objective

Systemic sclerosis (SSc; scleroderma) is characterized by a unique widespread vascular involvement that can lead to severe digital ischemia, pulmonary arterial hypertension (PAH), or other organ dysfunction. Microthrombotic events and procoagulation factors such as anti–β2-glycoprotein I (anti-β2GPI) or anticardiolipin antibodies (aCL) may be implicated in the development of these manifestations. This study was undertaken to investigate whether anti-β2GPI and aCL are correlated with macrovascular disease, including ischemic digital loss and PAH, in SSc patients.

Methods

Seventy-five SSc patients with a history of ischemic digital loss and 75 matched SSc controls were evaluated. Anticentromere antibodies (ACAs), anti-β2GPI, and aCL were measured, and clinical associations were determined using conditional and simple logistic regression models.

Results

Positivity for anti-β2GPI was significantly more frequent in SSc patients with digital loss than in patients without digital loss (P = 0.017), with the IgA isotype of anti-β2GPI showing the strongest association (odds ratio [OR] 4.0). There was no significant difference in aCL frequency between patients with digital loss and control patients. After adjustment for demographic characteristics, disease type, smoking, and ACA, anti-β2GPI positivity was significantly associated with active digital ischemia (OR 9.4), echocardiographically evident PAH (OR 4.8), and mortality (OR 2.9). ACA positivity was associated with history of digital loss (OR 3.28), but not with PAH or mortality. History of digital loss was strongly associated with increased mortality (OR 12.5).

Conclusion

Anti-β2GPI is significantly associated with macrovascular disease in SSc and independently predicts mortality. It is unclear whether it has a pathogenetic role or simply reveals the presence of underlying endothelial injury. The use of anti-β2GPI as a biomarker of vascular disease in SSc should be further explored.

Systemic sclerosis (SSc; scleroderma) is a multisystem disease characterized by immune activation, tissue fibrosis, and underlying vascular disease (1). These pathogenetic hallmarks are closely associated with one another and likely interact to ultimately determine the clinical phenotype expressed in the scleroderma patient. Microvascular disease is universally present and is characterized by structural damage (obliterative vasculopathy) as well as functional disturbances (secondary Raynaud's phenomenon [RP]). In a distinct subset of SSc patients, the disease presents with episodes of progressive digital ischemia, sometimes resulting in severe outcomes such as digital gangrene and amputation. This clinical presentation is usually associated with narrowing and occlusion of the ulnar (less frequently, radial), palmar, and digital arteries (2). Medium-sized and large arteries (macrovascular disease) can be affected in the lower extremities as well (3–5). Pulmonary arterial hypertension (PAH) also develops, with evidence of a progressive vascular disease with luminal narrowing and intimal thickening of medium-sized pulmonary arteries. Other macrovascular manifestations, such as coronary artery disease and cerebral vascular ischemia, have not been thoroughly studied in SSc, but current data suggest that they are not more prevalent in SSc patients than in the general population (3, 6).

Angiographic and pathologic studies indicate that the vascular disease in SSc is characterized by progressive obliteration of affected arteries, with defective angiogenesis and vasculogenesis resulting in extensive disease and inadequate collateral circulation. Endothelial dysfunction, vascular smooth muscle cell activation, and intimal hyperplasia characterize the vasculopathy in SSc (7). The occurrence of (micro)thrombotic events has also been linked to the onset of ischemic complications in SSc. Although autoantibodies directed against the endothelial surface are detected in SSc and their presence is associated with severe digital ischemia, no specific autoantigenic determinant has been consistently characterized (8).

Antiphospholipid antibodies (aPL) are immunoglobulins associated with recurrent thromboembolic events in primary antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). They are directed against negatively charged phospholipid-binding proteins that are mostly involved in blood coagulation. Clinical manifestations of APS were initially linked to the presence of anticardiolipin antibodies (aCL) and lupus anticoagulant (9). Subsequently, β2-glycoprotein I (β2GPI) was identified as the major target antigen for aCL or lupus anticoagulant (10). Anti-β2GPI antibodies are now included in the classification criteria for APS (11).

Although a causal association between anti-β2GPI and thrombotic events has been demonstrated in APS and SLE, the significance of this antibody in SSc and its relationship with the severity of clinical manifestations has not been fully addressed. The reported prevalence of anti-β2GPI in SSc ranges between 5% and 41% of patients (12–19). A similar prevalence range of aCL in SSc (12–45%) has been reported (13–15, 17–21). In none of these studies was the presence of aPL in SSc patients associated with the clinical manifestations typically seen in APS, such as recurrent arterial and venous thrombosis or pregnancy loss. Moreover, partly due to the low prevalence of aPL in SSc, most of these studies did not fully address or did not identify a correlation with any specific clinical manifestations. However, 2 studies demonstrated an association between overall aPL positivity or combined aCL/anti-β2GPI positivity and PAH, digital ischemia, or severe RP (12, 19). Interestingly, we have encountered several scleroderma patients with recent episodes of critical digital ischemia or digital loss who also tested positive for anti-β2GPI.

In the present study, we examined whether the prevalence of anti-β2GPI is increased among SSc patients with a history of digital loss. In addition, we investigated whether the presence of this autoantibody is associated with other clinical features of vascular disease, including active digital ischemia and pulmonary hypertension, as well as mortality.

PATIENTS AND METHODS

Patients.

Seventy-five SSc patients with a history of ischemic digital loss were identified in the Johns Hopkins Scleroderma Center database and matched by age, sex, race, and disease subtype with 75 SSc controls who had no history of ischemic digital loss. Patients with traumatic or postinfectious digital loss were excluded from the study. All patients met the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for SSc (22) and were classified as having diffuse cutaneous SSc or limited cutaneous SSc on the basis of the extent of skin involvement (23). Written informed consent was previously obtained (at the time of sample collection) from all patients. The present study was approved by the Johns Hopkins Institutional Review Board.

Clinical phenotyping.

Demographic and clinical data, including age, sex, ethnicity, education, smoking status, disease duration (calculated from the date of onset of first non-RP symptom), scleroderma subtype, specific organ involvement, and autoantibody status, were previously obtained on each patient at the time of the clinical visit. Ischemic digital loss was defined as amputation of a portion of or the entire finger or toe following an irreversible ischemic event at any time in the disease course. RP activity and presence of digital ischemia were determined using a previously published severity score (24) (0 = no RP, 1 = RP with or without vasodilator required, 2 = digital pitting scars, 3 = digital tip ulcerations, 4 = digital gangrene). Active digital ischemia was defined as an RP severity score of ≥3. Skin involvement was scored according to the modified Rodnan skin thickness score (MRSS [range 0–51]) (25). Internal organ involvement was assessed using previously published criteria (24). Pulmonary involvement was determined based on abnormal findings on pulmonary function tests (PFTs) (forced vital capacity [FVC] and single-breath diffusing capacity for carbon monoxide [DLCO], measured as the absolute value as well as the percent predicted value for race, sex, and age, according to the American Thoracic Society recommendations [26]). For the purpose of this study, a patient was considered to have evidence of PAH if the estimated right ventricular systolic pressure (eRVSP) determined by Doppler echocardiography was ≥40 mm Hg in separate tests and there was no overt clinical evidence of congestive heart failure, thromboembolic disease, or severe pulmonary interstitial fibrosis (FVC <50%). This assumption has been supported and confirmed in other studies (27). In addition, although information from right heart catheterization was limited in our data set, 16 of 18 SSc patients with a measured mean pulmonary artery pressure of >25 mm Hg, diagnostic of PAH, also had an eRVSP of >40 mm Hg on echocardiography. Heart, gastrointestinal, renal, or musculoskeletal involvement was considered present when the relative Medsger severity score (24) was ≥1. Evidence of sicca complex was determined by clinical criteria.

The medical record of each patient was also reviewed to identify previous manifestations of APS, including venous or arterial thrombosis. Information about pregnancy morbidity, thrombocytopenia, or livedo reticularis was not consistently available and was therefore omitted from the analysis.

Enzyme-linked immunosorbent assays (ELISAs).

Serum samples were previously obtained during routine clinical visits at the Johns Hopkins Scleroderma Center and were stored at −80°C. Levels of anti-β2GPI and aCL (IgM, IgA, and IgG isotypes) were quantitated using commercially available kits (Quanta LiteMicrowell ELISA) according to the instructions of the manufacturer (Inova Diagnostics, San Diego, CA). Results were converted into units using the standard calibrators provided. Samples with autoantibody values of >20 units were considered positive for all anti-β2GPI isotypes. This cutoff value for the assays was previously established by the manufacturer after testing sera from healthy controls and using the 95th percentile of the control values. The precision and reproducibility of the IgA and IgM anti-β2GPI assays were assessed at 3 levels of antibody activity (negative, low positive, and high positive) using patient sera. Intra- and interassay coefficients of variation were calculated after running each sample 5 times on 5 consecutive days; these data are summarized in Table 1. To further confirm the specificity of the assays, sera from 20 healthy donors were tested for anti-β2GPI (IgA, IgM, and IgG), with negative results (Figure 1).

Table 1. Summary of IgA and IgM anti-β2GPI assay precision*
SampleIntraassay, mean ± SD units (CV)Interassay, mean ± SD units (CV)Overall, mean ± SD units (CV)
  • *

    Each sample (negative, low positive, and high positive for IgA and IgM anti–β2-glycoprotein I antibody [anti-β2GPI]) was run 5 times (intraassay) on 5 consecutive days (interassay). Coefficients of variation (CVs) are percentages.

IgA anti-β2GPI   
 Negative6.1 ± 0.2 (2.9)5.8 ± 0.2 (3.0)5.9 ± 0.5 (7.9)
 Low positive29 ± 0.8 (2.7)31.8 ± 2.7 (8.4)31.8 ± 2.6 (8.1)
 High positive125 ± 1.4 (1.1)127 ± 3.3 (2.6)127 ± 4.9 (3.9)
IgM anti-β2GPI   
 Negative3.5 ± 0.2 (6.3)3.9 ± 0.4 (10.2)3.8 ± 0.4 (11.6)
 Low positive33.8 ± 1.1 (3.4)33 ± 2.7 (8.3)33 ± 2.9 (8.8)
 High positive216.3 ± 4.2 (1.9)214.9 ± 10 (4.7)214.9 ± 10.8 (5.1)
Figure 1.

A, Serum levels of IgM, IgA, and IgG anti–β2-glycoprotein I (anti-β2GPI) antibodies in systemic sclerosis patients with (n = 75) and those without (n = 75) digital loss and in healthy controls (n = 20). Horizontal bars show the means. Dotted line shows the cutoff value for positivity. B, Relative variation of IgM and IgA anti-β2GPI antibody titers from baseline (defined as 100%) over time. Each line represents a study patient from whom serially obtained samples were available (16 patients studied serially for IgM anti-β2GPI and 6 for IgA anti-β2GPI).

Statistical analysis.

Variables were transformed when a non-normal distribution was evident. The dependence of digital loss (response variable) on the risk factors considered (explanatory variables) was evaluated in a conditional logistic regression model. The association between autoantibody (anti-β2GPI and aCL) status (dichotomous) and disease characteristics or outcome was estimated using unadjusted conditional logistic regression for digital loss and an adjusted logistic regression model for digital ischemia, eRVSP, and mortality. Age, disease duration, skin score, PFT results, and eRVSP, together with RP and gastrointestinal and lung severity scores, were treated as continuous variables. The other sociodemographic or disease characteristics and autoantibody status were included in the models as dichotomous or categorical variables. Significance was tested using the regression coefficients, and the association between risk factors and outcome was expressed as the odds ratio (OR) and the corresponding 95% confidence interval (95% CI) or as a P value (considered significant when less than or equal to 0.05). Differences between anti-β2GPI isotype levels in patients with and those without digital loss were evaluated by rank sum test. Statistical analyses were performed with Stata, version 10 (StataCorp, College Station, TX).

RESULTS

Association between history of digital loss and SSc disease characteristics.

The sociodemographic and disease characteristics of the SSc patients with digital loss and the SSc controls (matched for age, sex, race, and disease subtype) are summarized in Table 2. There was no statistically significant difference between the 2 groups in terms of disease duration, presence of sicca symptoms, or gastrointestinal or kidney involvement.

Table 2. Sociodemographic and disease characteristics of the SSc patients, by history of digital loss*
VariableDigital loss (n = 75)No digital loss (n = 75)POR (95% CI)
  • *

    Except where indicated otherwise, values are the mean ± SD. SSc = systemic sclerosis; 95% CI = 95% confidence interval; NA = not applicable; FVC = forced vital capacity; DLCO = diffusing capacity for carbon monoxide; eRVSP = estimated right ventricular systolic pressure.

  • Based on a conditional logistic regression model for digital loss as a function of the sociodemographic or disease characteristic.

  • Time from first non–Raynaud's phenomenon (RP) symptom.

  • §

    Odds ratios (ORs) of digital loss are per unit increase in the continuous predictor.

  • Calculated using Kaplan-Meier survival estimates.

Age, years (range 22–86)52.5 ± 13.653.1 ± 12.5NA 
Female, % of patients7687NA 
Race/ethnicity, % of patients  NA 
 White7175  
 Black2423  
 Other52  
Smoking, % of patients  0.023 
 Never3760  
 Past3524 2.2 (1.0–4.7)
 Current2816 2.8 (1.1–7.2)
SSc type, % of patients  NA 
 Limited8385  
 Diffuse1715  
Disease duration, years (range 0.1–36.6)10.4 ± 7.99.6 ± 7.60.5361.0 (0.9–1.1)
Modified Rodnan skin score (0–51)8.0 ± 8.75.3 ± 6.80.0121.1 (1.0–1.2)
Gastrointestinal severity score (0–4)1.1 ± 0.91.4 ± 0.90.0840.7 (0.5–1.1)
Kidney involvement, % of patients12110.7961.1 (0.4–3.1)
Sicca complex, % of patients48560.2770.7 (0.3–1.4)
Lung severity score (0–4)1.8 ± 1.61.2 ± 1.20.0031.5 (1.1–2.0)
Pulmonary function§    
 FVC, % predicted82.1 ± 19.286.1 ± 18.00.2270.99 (0.97–1.01)
 DLCO, % predicted61.5 ± 17.671.9 ± 20.20.0020.97 (0.96–0.99)
Elevated eRVSP (>40 mm Hg), % of patients43320.1681.6 (0.8–3.2)
RP severity score (0–4)2.6 ± 0.81.5 ± 0.8<0.0013.4 (2.0–5.8)
Active digital ischemia (RP severity score >3), % of patients5515<0.0016.0 (2.5–14.2)
Mortality    
 Deceased, % of patients4811<0.00112.5 (3.0–52.8)
 Survival from SSc diagnosis, median years14.530.5<0.001 
Autoantibodies, % of patients    
 Anti–Scl-701180.5261.5 (0.4–5.3)
 Anticentromere63410.0033.3 (1.4–7.7)

In the digital loss group, the skin score, albeit low overall, was higher (P = 0.012) and the lung severity score was worse (P = 0.003), corresponding also to a significantly lower DLCO (P = 0.002) and a trend toward a higher eRVSP. Anticentromere antibody (ACA) positivity was more prevalent in the digital loss group (P = 0.003), but positivity for anti–Scl-70 was not. Significant differences between the 2 groups were noted in terms of RP severity score (P < 0.001), presence of active digital ischemia (P < 0.001), median length of survival from diagnosis (14.5 years versus 30.5 years; P < 0.001), and mortality (48% versus 11%; P < 0.001). Not surprisingly, past smoking (OR 2.2 [95% CI 1.0–4.7]), current smoking (OR 2.8 [95% CI 1.1–7.2]), more severe RP (OR 3.4 [95% CI 2.0–5.8]), and active digital ischemia (OR 6.0 [95% CI 2.5–14.2]) were significantly associated with history of digital loss. Importantly, we estimated that the odds of death were 12.5 times greater in the digital loss group compared with the SSc controls (OR 12.5 [95% CI 3.0–52.8]), and this was independent of disease duration. No significant history of arterial or venous thrombosis was identified by chart review in either group.

Antiphospholipid antibody profiles.

The prevalence of anti-β2GPI and aCL and their isotypes in cases and controls is shown in Table 3. Overall, anti-β2GPI was significantly more frequent in SSc patients with digital loss compared with SSc controls (P = 0.017), but aCL was not (P = 0.08). Anti-β2GPI was detected in 36% of patients and 19% of controls. The individual IgM and IgA anti-β2GPI isotypes occurred most frequently, while the IgG isotype was detected in only 1 patient, and the frequency of IgA anti-β2GPI was higher in SSc cases compared with SSc controls (P = 0.016). The distribution and combination of the different isotypes did not differ between the 2 groups; interestingly, however, in the majority of anti-β2GPI–positive SSc patients the IgM isotype was present alone. The prevalence of the IgA isotype was greater among anti-β2GPI–positive African American patients compared with white patients (50% versus 40%), but the difference was not statistically significant (P = 0.63). The analytical values for anti-β2GPI and aCL in study subjects and healthy controls are shown in Figure 1A. Positive autoantibody isotypes displayed a moderate to high titer, but their mean levels did not differ significantly between patients with and those without digital loss.

Table 3. Frequency and isotype distribution of anti-β2GPI and aCL in the SSc patients, by history of digital loss*
 Digital loss (n = 75)No digital loss (n = 75)P
  • *

    Values are the number (%). Anti-β2GPI = anti–β2-glycoprotein I antibody; aCL = anticardiolipin antibody; SSc = systemic sclerosis; NA = not applicable.

  • Calculated using a conditional logistic regression model.

Anti-β2GPI   
 Any positive27 (36)14 (19)0.017
 IgM20 (27)11 (15)0.08
 IgA13 (17)4 (5)0.016
 IgG1 (1)0 (0)NA
 Isotype distribution   
  IgM only13 (17)9 (12)0.356
  IgA only7 (9)2 (3)0.086
  IgG only0 (0)0 (0)NA
  IgM + IgA5 (7)2 (3)0.246
  IgM + IgA + IgG1 (1)0 (0)NA
aCL   
 Any positive18 (24)10 (13)0.08
 IgM14 (19)9 (12)0.248
 IgA3 (4)0 (0)NA
 IgG7 (9)2 (3)0.097
 Isotype distribution   
  IgM only10 (13)8 (11)0.615
  IgA only1 (1)(0)NA
  IgG only3 (4)1 (1)NA
  IgM + IgA(0)(0)NA
  IgM + IgG2 (3)1 (1)NA
  IgM + IgA + IgG2 (3)(0)NA

Serially obtained serum samples were available from a small number of patients. We observed that all patients who were initially found to be positive for anti-β2GPI remained positive over time. Titers of IgA anti-β2GPI seemed to remain more stable, while titers of IgM anti-β2GPI exhibited greater fluctuation (Figure 1B). In addition, IgM anti-β2GPI did not undergo isotype class switching. We observed only 1 seroconversion from anti-β2GPI negative to positive (IgM).

Association of aPL with SSc disease characteristics.

Data on SSc cases and controls were combined to assess the association between positivity for aPL and SSc disease characteristics. With the exception of older age in anti-β2GPI–positive SSc patients (mean ± SD 55.1 ± 15.9 years versus 52 ± 11.7 years; P = 0.008), sociodemographic variables and general disease characteristics were similar in the 2 groups for both autoantibodies (anti-β2GPI and aCL) (data not shown). Table 4 summarizes the associations of anti-β2GPI and aCL positivity with digital loss (conditional logistic regression) and with digital ischemia, eRVSP (≥40 mm Hg), and mortality (logistic regression models). Anti-β2GPI exhibited a significant association with digital loss (OR 2.4 [95% CI 1.1–5.3]) and other features of macrovascular disease, as well as mortality, in SSc patients. Of note, the associations were maintained after adjustment for age, sex, race, disease type, smoking status, digital loss, and ACA status (OR 9.4 [95% CI 3.5–25.4] for active digital ischemia, OR 4.8 [95% CI 1.0–11.4] for elevated eRVSP, and OR 2.9 [95% CI 1.1–7.7] for mortality), indicating that anti-β2GPI is independently associated with these features. Positive trends, but no statistically significant associations, were noted in relation to aCL status.

Table 4. Associations of anti-β2GPI and aCL positivity with clinical features of macrovascular disease and mortality in the SSc patients*
 Feature or mortality presentFeature or mortality absentUnadjusted OR (95% CI)Adjusted OR (95% CI)
  • *

    Values are the number (%); n values are the number of systemic sclerosis (SSc) patients in whom the feature was present/absent. Anti-β2GPI = anti–β2-glycoprotein I antibody; aCL = anticardiolipin antibody; OR = odds ratio; 95% CI = 95% confidence interval; NA = not applicable.

  • Based on unadjusted conditional logistic regression (for digital loss) or on logistic regression models (for digital ischemia, estimated right ventricular systolic pressure [eRVSP], and mortality).

  • Adjusted for age, sex, race, disease type, smoking status, and anticentromere antibody status.

  • §

    Status not known for 13 patients.

Digital loss (n = 75/75)    
 Anti-β2GPI    
  Any positive27 (36)14 (19)2.4 (1.1–5.3) 
  IgM20 (27)11 (15)2.0 (0.9–4.5) 
  IgA13 (17)4 (5)4.0 (1.1–14.2) 
  IgG1 (1)0 (0)NA 
 aCL    
  Any positive18 (24)10 (13)2.1 (0.9–5.3) 
  IgM14 (19)9 (12)1.7 (0.7–4.4) 
  IgA3 (4)0 (0)NA 
  IgG7 (9)2 (3)6.0 (0.7–49.8) 
Active digital ischemia (n = 52/98)    
 Anti-β2GPI    
  Any positive26 (50)15 (15)5.5 (2.6–12.0)9.4 (3.5–25.4)
  IgM20 (38)11 (11)4.9 (2.1–11.4)13.9 (4.4–43.5)
  IgA12 (23)5 (5)5.6 (1.8–16.9)4.5 (1.4–15.3)
  IgG0 (0)1 (1)NANA
 aCL    
  Any positive11 (21)17 (17)1.3 (0.5–3.0)1.2 (0.5–3.3)
  IgM10 (19)13 (13)1.6 (0.6–3.8)1.7 (0.6–4.9)
  IgA3 (6)0 (0)NANA
  IgG3 (6)6 (6)0.9 (0.2–3.9)0.5 (0.1–2.8)
eRVSP >40 mm Hg (n = 56/94)    
 Anti-β2GPI    
  Any positive27 (48)14 (15)5.3 (2.5–11.5)4.8 (1.0–11.4)
  IgM21 (38)10 (11)5.0 (2.2–11.8)5.4 (2.1–14.4)
  IgA12 (21)5 (5)4.9 (1.6–14.6)4.6 (1.3–15.7)
  IgG1 (2)0 (0)NANA
 aCL    
  Any positive15 (27)13 (14)2.3 (1.0–5.2)1.7 (0.7–4.1)
  IgM12 (21)11 (12)2.1 (0.8–5.0)1.6 (0.6–4.2)
  IgA3 (5)0 (0)NANA
  IgG7 (13)2 (2)6.6 (1.3–32.9)4.1 (0.7–22.4)
Death (n = 39/98)§    
 Anti-β2GPI    
  Any positive17 (44)23 (23)2.5 (1.1–5.5)2.9 (1.1–7.7)
  IgM12 (31)17 (17)2.0 (0.86–4.7)3.1 (1.1–9.2)
  IgA9 (23)7 (7)3.9 (1.3–10.9)2.8 (0.8–9.6)
  IgG0 (0)1 (1)NANA
 aCL    
  Any positive10 (26)14 (14)2.0 (0.8–5.0)2.2 (0.8–6.3)
  IgM9 (23)11 (11)2.3 (0.8–6.0)2.8 (0.9–8.6)
  IgA3 (8)0 (0)NANA
  IgG3 (8)5 (5)1.5 (0.3–6.5)1.1 (0.2–6.8)

Using the adjusted logistic regression model, data were also analyzed to identify other predictors of higher mortality (beyond history of digital loss or anti-β2GPI status). Comparing patients who were still living versus deceased patients, significant associations with current smoking status (OR 4.6 [95% CI 1.6–13.6], P = 0.005), active digital ischemia (OR 5.8 [95% CI 2.3–14.9], P < 0.001), and skin score (mean ± SD MRSS 9.6 ± 10.6 versus 5.2 ± 6.3; P = 0.005) were found. Disease duration, anti–Scl-70 status, ACA status, elevated eRVSP, and the other sociodemographic and disease characteristics were comparable in the 2 groups.

Finally, the relationship between anti-β2GPI and ACA status was explored. Forty-nine patients were positive for ACA only, and 29 had both anti-β2GPI and ACA. As summarized in Table 5, the presence of anti-β2GPI carried, independent of ACA status, a significant association with increased risk of active digital ischemia (OR 16.4 [95% CI 3.4–80.5], P < 0.001), elevated eRVSP suggestive of PAH (OR 7.9 [95% CI 2.6–24.4], P = 0.002), and mortality (OR 2.9 [95% CI 1.1–10.1], P = 0.004).

Table 5. Association of digital loss, digital ischemia, eRVSP, and mortality with the presence of anti-β2GPI and ACAs*
 All ACA+ (n = 78)ACA+ and anti-β2GPI+ (n = 29), no. (%)ACA+ only (n = 49), no. (%)ACA+ and anti-β2GPI+ vs. ACA+ only, OR (95% CI)
No. (%)OR (95% CI)
  • *

    Odds ratios (ORs) for digital loss were calculated using conditional logistic regression. ORs for digital ischemia, estimated right ventricular systolic pressure (eRVSP), and mortality were calculated using logistic regression with adjustment for age, sex, race, disease type, digital loss, and smoking status. Anti-β2GPI = anti–β2-glycoprotein I antibody; ACA = anticentromere antibody; 95% CI = 95% confidence interval.

Digital loss47 (60)3.28 (1.4–7.7)19 (66)28 (57)1.5 (0.5–4.8)
Digital ischemia27 (35)1.15 (0.3–3.9)17 (59)10 (20)16.4 (3.4–80.5)
High eRVSP33 (42)1.3 (0.4–3.8)20 (69)13 (27)7.9 (2.6–24.4)
Mortality22 (28)0.4 (0.1–1.3)11 (38)10 (20)2.9 (1.1–10.1)

DISCUSSION

This investigation addressed the association between the presence of aPL and vascular disease in a large, well-characterized cohort of SSc patients. Our study showed that anti-β2GPI is more prevalent in SSc patients with digital loss and is significantly associated with features of macrovascular disease, including active digital ischemia and echocardiographically evident PAH. Also, we report for the first time that positivity for anti-β2GPI is independently associated with higher mortality.

We found that patients with a history of ischemic digital loss have, independent of disease duration, a substantially increased risk of death (OR 12.5), together with worse lung severity scores and lower DLCO. Based on these associations, it is possible that pulmonary vascular disease may be partially responsible for the worse outcome. In fact, at the time of sample testing, the eRVSP was higher overall in patients with digital loss. Smoking (current or past) was also more prevalent in the digital loss group and may also have contributed to the more severe vasculopathy.

We confirmed the known association between ACAs and vascular disease in SSc, and in particular, with severe digital ischemia and digital loss (28, 29). However, in accordance with previously reported results (30), we found that ACA status did not independently predict higher mortality in SSc patients.

Anti-β2GPI was significantly more frequent in SSc patients with digital loss compared with SSc controls (36% versus 19%; P = 0.017). We detected almost exclusively IgM and IgA isotypes, alone or in combination. Similar to published data on patients with APS (31), the IgA isotype was more prevalent in anti-β2GPI–positive African American patients, although the difference was not statistically significant. In contrast to previous studies (12–19), all of the anti-β2GPI isotypes, including IgA, were measured in our SSc patients. In several of them, IgA was found to be the only positive isotype present, and was found at relatively high titers. The exclusion of IgA anti-β2GPI measurement by other investigators may have been driven by the suggestion in some of the older literature that screening for IgA anti-β2GPI is not helpful for diagnosis of APS in SLE (32, 33). This issue remains slightly controversial, with more recent publications reporting a significant association between IgA aPL and thrombosis (34, 35).

Importantly, anti-β2GPI, and in particular the IgA isotype, has been associated with higher risk of other, non–connective tissue disorder–related, vascular conditions, including peripheral vascular disease, cerebral ischemia, and myocardial infarction (36–39). Franck et al reported that the IgA isotype of aCL and anti-β2GPI was associated with peripheral arterial disease, with an adjusted OR of 12.1 (95% CI 5.8–30) (36). The same group and other authors (37) showed that IgA anti-β2GPI was significantly more frequent in patients with ischemic stroke. Meroni and colleagues found that IgM and IgG anti-β2GPI (IgA not tested) carry a higher risk of myocardial infarction in young premenopausal women, independent of underlying atherosclerotic disease (38). Other investigators detected higher anti-β2GPI levels in patients with acute coronary syndrome compared with controls (14.4% versus 2%), with particular significance for the IgA isotype (39). All of these studies consistently linked different clinical manifestations of larger vessel disease with the detection of higher levels of anti-β2GPI, in particular of the IgA isotype. Our results extend these findings to vascular manifestations associated with SSc and suggest that anti-β2GPI represents an independent biomarker in SSc macrovascular disease.

Previous studies of the prevalence and significance of anti-β2GPI in SSc are limited (11–18), and evidence of APS clinical features in anti-β2GPI–positive SSc patients was not detected in any of those investigations. Only 2 studies showed a significant association between aPL and SSc vascular disease manifestations, such as isolated PAH, peripheral ischemia, and digital pitting (12, 19).

While we found that aCL was more frequent in patients with digital loss (24% versus 13%), the association did not reach statistical significance (P = 0.08). Similarly, Herrick et al found no significant association between presence of IgM and IgG aCL (IgA not studied) and severe ischemia or amputation in SSc patients (29). Also, consistent with the majority of previous reports, we did not detect any association between aCL and clinical features of APS or macrovascular disease in our cohort. In contrast, after correcting for confounders or disease modifiers, we showed a strong, statistically significant association between anti-β2GPI positivity and SSc manifestations of macrovascular disease, such as digital loss, active digital ischemia, and echocardiographically evident PAH.

The independent association of anti-β2GPI positivity with mortality (OR 2.9 [95% CI 1.1–7.7]) is another important finding that emerged from our study. We speculate that anti-β2GPI positivity identifies SSc patients with persistent and widespread vascular disease. Our data support the possibility that the detection of these autoantibodies in patients with active digital ischemia or PAH may indicate sustained and progressive endothelial/vascular damage and predict worse outcome. Prospective studies to address this initial observation are under way.

How the immune response against β2GPI is generated, and what role anti-β2GPI plays in SSc vascular manifestations, is not known. Beta2-GPI is an abundant plasma protein that can be expressed on the surface of endothelial cells and has been found in the intima of the arterial wall as well as within atherosclerotic plaques (40, 41). The formation of complexes with oxidized low-density lipoprotein or anionic phospholipids has been shown to modify and increase the immunogenicity of β2GPI, facilitating antigen presentation by macrophages and dendritic cells (42). Beta2-GPI–reactive T cells have been identified in the peripheral blood and within atherosclerotic plaques, further confirming that this protein can become immunogenic under certain circumstances (43, 44). On the other hand, anti-β2GPI is present also in younger SSc patients without overt evidence of atherosclerosis, and can be found in association with acute vascular events, suggesting that other mechanisms, such as ongoing perturbation of endothelium homeostasis, may be relevant for their generation.

The dominant anti-IgM/IgA β2GPI response detected in SSc sera represents a clear distinction from the anti-β2GPI specificities usually observed in patients with APS and SLE, typically characterized by a substantial IgG isotype class switch (32). This was not observed in the analysis conducted on serially obtained serum samples from several IgM anti-β2GPI–positive patients in the present study. If confirmed, this interesting finding may suggest that the generation of anti-β2GPI in SSc occurs through a T cell–independent mechanism. Molecular mimicry between microbial pathogens and β2GPI epitopes has also been identified by some authors (45). Many of these agents (i.e., Helicobacter pylori, adenovirus) are involved in mucosal infections and may trigger an immune response biased toward production of IgA anti-β2GPI. The influence of transforming growth factor β should also be considered. This cytokine exerts profibrotic effects in SSc and can promote IgA class switching and plasma cell IgA secretion (46, 47).

Anti-β2GPI antibodies can contribute to a prothrombotic status through many mechanisms, for example, by modifying the functional properties of thrombin and protein C (48, 49). They also have the ability to bind to negatively charged phospholipids on the surface of activated or apoptotic cells, promoting proinflammatory responses (50). Whether anti-β2GPI exerts some pathogenetic role in SSc by means of its antiendothelial and procoagulation properties, or is merely an epiphenomenon of the ongoing vascular damage/dysfunction, still remains to be defined. Autoantibodies directed against other proteins (i.e., annexin V) binding to negatively charged phospholipids and interfering with the coagulation cascade have also been detected in SSc (51). However, their role in the pathogenesis of SSc vascular manifestations has never been confirmed.

Strengths of the present study include the relatively large number of patients, their excellent clinical phenotyping, the use of commercial ELISA kits widely used in hospital clinical laboratories, and a careful statistical approach, allowing us to properly interrogate the clinical significance of aCL and anti-β2GPI in SSc. However, there are some limitations. The case–control and cross-sectional study design does not account for variation in serum levels of aPL over time. Lupus anticoagulant testing was not performed due to the limited availability of plasma samples. Data regarding the use of platelet antiaggregants and anticoagulants were not included in the study, and such therapies may have influenced the long-term survival in our patients.

In conclusion, our investigation showed significant associations between anti-β2GPI positivity and clinical features of severe vascular disease in SSc, including active digital ischemia, digital loss, and echocardiographic evidence of PAH. An independent association with higher mortality was also demonstrated. These findings indicate that anti-β2GPI should be further investigated as a reproducible biomarker of vascular disease and a predictor of clinical outcomes in SSc.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Boin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Boin, Franchini, Rosen, Wigley, Casciola-Rosen.

Acquisition of data. Boin, Franchini, Casciola-Rosen.

Analysis and interpretation of data. Boin, Rosen, Wigley, Casciola-Rosen.

Acknowledgements

The authors thank Brandon Boring and David Hines for their valuable technical assistance.

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