Jane E. Salmon

A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to autoimmune disease.

A novel polymorphism in the extracellular domain 2 (EC2) of FcgammaRIIIA affects ligand binding by natural killer (NK) cells and monocytes from genotyped homozygous normal donors independently of receptor expression. The nonconservative T to G substitution at nucleotide 559 predicts a change of phenylalanine (F) to valine (V) at amino acid position 176. Compared with F/F homozygotes, FcgammaRIIIa expressed on NK cells and monocytes in V/V homozygotes bound more IgG1 and IgG3 despite identical levels of receptor expression. In response to a standard aggregated human IgG stimulus, FcgammaRIIIa engagement on NK cells from V/V (high-binding) homozygotes led to a larger rise in [Ca2+]i, a greater level of NK cell activation, and a more rapid induction of activation-induced cell death (by apoptosis). Investigation of an independently phenotyped normal cohort revealed that all donors with a low binding phenotype are F/F homozygotes, while all phenotypic high binding donors have at least one V allele. Initial analysis of 200 patients with SLE indicates a strong association of the low binding phenotype with disease, especially in patients with nephritis who have an underrepresentation of the homozygous high binding phenotype. Thus, the FcgammaRIIIa polymorphism at residue 176 appears to impact directly on human biology, an effect which may extend beyond autoimmune disease characterized by immune complexes to host defense mechanisms.

Heparin prevents antiphospholipid antibody–induced fetal loss by inhibiting complement activation

The antiphospholipid syndrome (APS) is defined by thrombosis and recurrent pregnancy loss in the presence of antiphospholipid (aPL) antibodies and is generally treated with anticoagulation therapy. Because complement activation is essential and causative in aPL antibody–induced fetal injury, we hypothesized that heparin protects pregnant APS patients from complications through inhibition of complement. Treatment with heparin (unfractionated or low molecular weight) prevented complement activation in vivo and in vitro and protected mice from pregnancy complications induced by aPL antibodies. Neither fondaparinux nor hirudin, other anticoagulants, inhibited the generation of complement split products or prevented pregnancy loss, demonstrating that anticoagulation therapy is insufficient protection against APS-associated miscarriage. Our data indicate that heparins prevent obstetrical complications in women with APS because they block activation of complement induced by aPL antibodies targeted to decidual tissues, rather than by their anticoagulant effects.

Complement C3 Activation Is Required for Antiphospholipid Antibody-induced Fetal Loss

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, vascular thrombosis, and thrombocytopenia occurring in the presence of antiphospholipid (aPL) antibodies. The pathogenesis of fetal loss and tissue injury in APS is incompletely understood, but is thought to involve platelet and endothelial cell activation as well as procoagulant effects of aPL antibodies acting directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation in the placenta leads to fetal death in utero. We hypothesized that aPL antibodies activate complement in the placenta, generating split products that mediate placental injury and lead to fetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. We found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1–related gene/protein y (Crry)-Ig, blocks fetal loss and growth retardation. Furthermore, mice deficient in complement C3 were resistant to fetal injury induced by aPL antibodies. While antigenic epitopes recognized by aPL antibodies are important in the pathogenesis of APS, our data show that in vivo complement activation is required for aPL antibody-induced fetal loss and growth retardation.

Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans

Allelic variants of Fc gamma R confer distinct phagocytic capacities providing a mechanism for heritable susceptibility to immune complex disease. Human Fc gamma RIIa has two codominantly expressed alleles, R131 and H131, which differ substantially in their ability to ligate human IgG2. The Fc gamma RIIa-H131 is the only human Fc gamma R which recognizes IgG2 efficiently and optimal IgG2 handling occurs only in the homozygous state. Therefore, since immune complex clearance is essential in SLE, we hypothesized that Fc gamma RIIA genes are important disease susceptibility factors for SLE, particularly lupus nephritis. In a two-stage cross-sectional study, we compared the distribution of Fc gamma RIIA alleles in African Americans with SLE to that in African American non-SLE controls. A pilot study of 43 SLE patients and 39 controls demonstrated a skewed distribution of Fc gamma RIIA alleles, with only 9% of SLE patients homozygous for Fc gamma RIIa-H131 compared with 36% of controls (odds ratio, 0.18; 95% CI, 0.05-0.69, P = 0.009). This was confirmed with a multicenter study of 214 SLE patients and 100 non-SLE controls. The altered distribution of Fc gamma RIIA alleles was most striking in lupus nephritis. Trend analysis of the genotype distribution showed a highly significant decrease in Fc gamma RIIA-H131 as the likelihood for lupus nephritis increased (P = 0.0004) consistent with a protective effect of the Fc gamma RIIA-H131 gene. The skewing in the distribution of Fc gamma RIIA alleles identifies this gene as a risk factor with pathophysiologic importance for the SLE diathesis in African Americans.

Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgGs from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.

Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction

Immune mechanisms have been implicated in placental dysfunction in patients with recurrent miscarriages and intrauterine growth restriction (IUGR), but the mediators are undefined. Here we show that complement activation, particularly C5a, is a required intermediary event in the pathogenesis of placental and fetal injury in an antibody-independent mouse model of spontaneous miscarriage and IUGR, and that complement activation causes dysregulation of the angiogenic factors required for normal placental development. Pregnancies complicated by miscarriage or growth restriction were characterized by inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor 1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation in vivo blocked the increase in sVEGFR-1 and rescued pregnancies. In vitro stimulation of monocytes with products of the complement cascade directly triggered release of sVEGFR-1, which sequesters VEGF. These studies provide the first evidence linking the complement system to angiogenic factor imbalance associated with placental dysfunction, and identify a new effector of immune-triggered pregnancy complications.

Preclinical Carotid Atherosclerosis in Patients with Rheumatoid Arthritis

Context Patients with rheumatoid arthritis are prone to premature death from coronary heart disease despite few risk factors. Researchers have wondered if chronic inflammation is a trigger. Content The authors measured inflammatory markers and risk factors for coronary heart disease in 98 matched case-patients and controls (mean age, 48 years). Carotid ultrasonography revealed that 44% of case-patients and 15% of controls had atherosclerotic plaque. Independent predictors of plaque were age, smoking, and rheumatoid arthritis. In patients with rheumatoid arthritis, inflammatory mediators did not predict plaque. Limitations This cross-sectional study cannot prove that rheumatoid arthritis accelerates atherosclerosis. Interpretation Carotid atherosclerotic plaque is much more common in patients with rheumatoid arthritis than in controls. The mechanism remains unknown. The Editors Compared with the general population, patients with rheumatoid arthritis die prematurely (1, 2), primarily because of cardiovascular disease (1-3). Women with this disease have high rates of nonfatal myocardial infarction (4-6), even in the absence of traditional risk factors for atherosclerosis (4, 5, 7). Although markers of disease severity have been linked to an increase in overall mortality rates (1), researchers have not been able to clearly identify specific aspects of rheumatoid arthritis or its treatment that might heighten the risk for cardiovascular disease. Use of corticosteroids or disease-modifying antirheumatic drugs does not appear to increase the risk for cardiovascular events (2). In fact, a large longitudinal study recently reported that death rates from myocardial infarction among North American patients with rheumatoid arthritis had declined to the level seen in the general population (thereby yielding a greater magnitude of decline) in the setting of increased methotrexate use (8). In another U.S. study, methotrexate use was associated with lower all-cause mortality rates in rheumatoid arthritis, mostly because cardiovascular mortality rates were decreased (9). Early diagnosis of atherosclerosis in this population might trigger more aggressive prophylaxis, but we have not determined the prevalence of preclinical atherosclerosis or identified markers for the disease. In this study, we examined the prevalence of atherosclerosis in patients with rheumatoid arthritis by using ultrasonogram-defined carotid artery plaque as a direct measure and proxy for generalized atherosclerosis and as a surrogate for coronary atherosclerosis; we also examined those features of rheumatoid arthritis that predict plaque presence. Previous studies reported that plaque prevalence in rheumatoid arthritis is statistically similar to that of control populations (10, 11). However, in systemic lupus erythematosus, another autoimmune disease characterized by chronic inflammation, cross-sectional studies that were conducted by us and by others showed a marked increase in plaque compared with that seen in carefully matched controls (12, 13). The increased plaque rate in systemic lupus erythematosus is associated with chronic inflammation (not with treatment or with traditional atherosclerosis risk factors), suggesting that similar factors may be at work in rheumatoid arthritis. Preclinical disease may also be identified by using ultrasonography to determine carotid intimamedia thickness, an indirect measure of atherosclerosis. Intimamedia thickness was increased in 2 studies of East Asian patients with rheumatoid arthritis (14, 15) but not in a U.S. study (11). Intimamedia thickness varied more with disease duration (14, 15), but an association with serum C-reactive protein levels and erythrocyte sedimentation rate (2 markers of inflammation) has not been established because of conflicting reports (11, 15). Intimamedia thickness does not always correlate with atherosclerosis, particularly in relatively young individuals with chronic inflammatory disease (12, 13), and it may measure other aspects of vascular disease. Discrete atherosclerotic plaque is a potent independent predictor of incident cardiovascular disease, whereas intimamedia thickness in areas free of discrete plaque has limited value as a marker after traditional risk factors for cardiovascular disease are considered (16-18). Because of conflicting data regarding premature preclinical atherosclerosis in rheumatoid arthritis, we chose to use the direct measure of plaque to examine the prevalence of carotid atherosclerosis in consecutive unselected, nonhospitalized patients with rheumatoid arthritis and matched controls. For our other primary outcome, we sought to determine those clinical and biological measures that best predict the presence of plaque. Methods Study Sample We consecutively recruited patients who met the American College of Rheumatologys classification criteria for a diagnosis (possessing at least 4 of 7 criteria) of rheumatoid arthritis (19) and who were enrolled in the Rheumatoid Arthritis Registry at the Hospital for Special Surgery in New York. Patients were recruited at regular visits with their rheumatologists during a 15-month period (participation rate, 94%). Exclusion criteria were age younger than 18 years, serum creatinine level of 270 mol/L or greater (3.0 mg/dL) or creatinine clearance of 0.50 mL/s or less (30 mL/min), or current or recent (within the past 3 months) pregnancy. We quantified extent of disease by recording extra-articular manifestations (for example, the Sjgren syndrome, leg ulcers, and evidence of vasculitis, such as nail fold infarcts, splinter hemorrhages, and motor neuropathy), active joint count (number of tender or swollen joints), number of joints irreversibly damaged (fixed deformity or surgical replacement) (20), and the patients score on the Multidimensional Health Assessment Questionnaire (21). We recorded treatment by patient interview and chart review. Because treatment is often intermittent or at varying dosages, we tabulated medication use as never, former, or current. An 83-year-old woman had a previous stroke that was documented by magnetic resonance imaging, and a 45-year-old man had had coronary artery bypass surgery; we calculated our results both including and excluding these 2 patients. Patients were matched to controls on the basis of age (within 5 years), sex, and ethnicity. Controls were normotensive and hypertensive individuals who participated in longitudinal studies funded by the National Institutes of Health (22, 23) at the Hypertension Center of The New York Hospital and who underwent similar imaging protocols. We assessed patients for traditional cardiovascular disease risk factors: family history of myocardial infarction in first-degree male relatives younger than 55 years of age or first-degree female relatives younger than 65 years of age, smoking, hypertension (defined as blood pressure of 140/90 mm Hg or higher or the prescribed use of antihypertensive medications), diabetes mellitus (self-reported diagnosis), and fasting serum cholesterol levels. Hypertensive controls were studied after antihypertensive medications had been withheld for 3 or more weeks, whereas antihypertensive medications were not systematically withheld in hypertensive patients with rheumatoid arthritis. Brachial blood pressure was obtained at the end of the ultrasonographic studies after patients had remained in the supine position for 45 to 60 minutes in a quiet, darkened room. Of 100 patients with rheumatoid arthritis, a 74-year-old man was unable to be matched to a suitable control and a woman was excluded because she met diagnostic criteria for systemic lupus erythematosus. The institutional review board approved the study protocol, and all participants gave written informed consent. Carotid Ultrasonography All study participants underwent carotid ultrasonography, which was performed by experienced research sonographers who used an identical protocol. A single cardiologist, who was blinded to the identity of the study participants, interpreted the results. In brief, as previously described (22, 23), participants were studied in the supine position with slight hyperextension of the neck. Both extracranial carotid arterial systems were extensively scanned in multiple planes to optimize identification of atherosclerosis, which was defined as discrete plaque protruding into the lumen at least 50% beyond the diameter of the surrounding wall. Doppler interrogation was performed to evaluate the presence of significant (50% diameter reduction) obstruction. Intimamedia thickness was measured from end-diastolic (minimum dimension) M-mode images of the far wall of the distal common carotid artery. Intimamedia thickness was not measured in a location containing plaque. Mean values of right and left intimamedia thicknesses are presented. Reproducibility of intimamedia thickness and detection of plaque has been well documented (24-26). Carotid ultrasonographic studies were performed in the control group before 1999, whereas studies in the patients with rheumatoid arthritis were performed between 2000 and 2002. Laboratory Assessment Laboratory assessment of the patients with rheumatoid arthritis included routine chemistries and serum rheumatoid factor level. A high-sensitivity assay to determine serum levels of C-reactive protein was analyzed with a Cobas Integra system (Roche Diagnostics, Basel, Switzerland). Serum lipoprotein(a) levels were measured with an immunoturbidometric reagent (Diasorin, Stillwater, Minnesota) on a Roche Diagnostics Cobas Fara II system. Serum interleukin-6 levels were measured by automated enzyme immunoassay (Biosource International, Camarilo, California) on a Roche Diagnostics Cobas Core II analyzer. Serum levels of soluble intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 were measured by enzyme-linked immunosorbent assay (Caltag, Burlingame, California, and R&D Systems, Minneapolis, Minnesota,

Allelic polymorphisms of human Fc gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in human phagocyte function.

Two different allelic polymorphisms among the isoforms of human Fc gamma receptors have been defined: the low-responder (LR)-high-responder (HR) polymorphism of huFc gamma RIIA expressed on both PMN and monocytes and the NA1-NA2 polymorphism of the neutrophil Fc gamma RIII (huFc gamma RIIIB). To address the issues of whether the LR-HR polymorphism has a significant impact on Fc gamma R-mediated functions in human blood cells and whether any differences in LR-HR might be related to higher Fc gamma R-mediated phagocytosis in NA1 donors, we examined Fc gamma R-specific binding and internalization by donors homozygous for the two huFc gamma RIIA alleles. PMN from LR homozygotes showed consistently higher levels of internalization of erythrocytes opsonized with pooled human IgG (E-hIgG). The absence of an LR-HR phagocytic difference with erythrocytes opsonized with either anti-Fc gamma RIIA MAb IV.3 or rabbit IgG, as opposed to E-hIgG, suggested that the Fc piece of the opsonin might be important for this LR-HR difference. Accordingly, we studied HR and LR homozygotes with human IgG subclass-specific probes. Both PMN (independent of huFc gamma RIIIB phenotype) and monocytes from LR donors bound and internalized erythrocytes coated with human IgG2 (E-hIgG2) efficiently, whereas phagocytes from HR donors did so poorly. E-hIgG2 internalization was completely abrogated by blockade of the ligand binding site of huFc gamma RIIA with IV.3 Fab, indicating that huFc gamma RIIA is essential for the handling of hIgG2 and that the mechanism of the LR-HR phagocytic difference is at the level of ligand binding to huFc gamma RIIA. In contrast, the difference in internalization of E-hIgG between NA1 and NA2 homozygous donors was independent of the huFc gamma RIIA phenotype and did not manifest differences in ligand binding. Thus, the two known allelic polymorphisms of human Fc gamma R have distinct and independent mechanisms for altering receptor function, which may influence host defense and immune complex handling.

Arterial Stiffness in Chronic Inflammatory Diseases

Chronic inflammatory diseases are associated with premature atherosclerosis; however, it is unknown whether arterial stiffness is increased in this setting, possibly as a manifestation of vascular disease preceding and/or independent of atherosclerosis. Carotid ultrasonography and radial applanation tonometry were performed in 101 patients with systemic lupus erythematosus, 80 patients with rheumatoid arthritis, and 105 healthy control subjects. The 3 groups were comparable in age, gender, and carotid artery absolute and relative wall thickness. Atherosclerotic plaque was more common in lupus (46%) and rheumatoid arthritis (38%) patients than in controls (23%) (P<0.003). Although control subjects had higher central and peripheral blood pressures, arterial stiffness was increased in patient groups compared with controls (lupus, rheumatoid arthritis, controls, respectively: &bgr;: 3.36 versus 3.22 versus 2.60, P<0.001; Young’s modulus: 441 versus 452 versus 366 mm Hg/cm, P=0.004; Peterson’s elastic modulus: 278 versus 273 versus 216 mm Hg, P<0.001) after adjustment for differences in mean brachial pressure. In multivariate analysis involving the entire population, arterial stiffness was independently related to age, serum glucose, and the presence of chronic inflammatory disease. In multivariate analysis restricted to the patients, arterial stiffness was independently related to age at diagnosis, disease duration, serum cholesterol, and C-reactive protein (and IL-6, when substituted for C-reactive protein). When analyses were repeated in the 186 study subjects without carotid plaque, arterial stiffness remained significantly elevated in patient groups after adjustment for differences in age and mean brachial pressure. In conclusion, arterial stiffness is increased in chronic inflammatory disorders independent of the presence of atherosclerosis and is related to disease duration, cholesterol, and the inflammatory mediator C-reactive protein and the cytokine that stimulates its production, IL-6.

Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines.

Immune complex-mediated inflammatory responses are initiated by FcγR on phagocytes. We report in this study that an inhibitory receptor, FcγRIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory FcγR it down-regulates effector function. FcγRIIb2 expression is increased by IL-4 and decreased by IFN-γ, in contrast to the activating receptor, FcγRIIa, which is increased by IFN-γ and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing FcγR systems, altering the balance of activating and inhibiting FcγR. The detection and cytokine modulation of FcγRIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease.