Systemic Lupus Erythematosus, Thrombocytopenia, Microangiopathic ...

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Thrombocytopenia in patients with acute systemic lupus erythematosus (SLE) frequently presents the ..... antibodies in thrombotic thrombocytopenic purpura. Br.
British Journal of Rheumatology 1997;36:794–798 GRAND ROUNDS IN RHEUMATOLOGY EDITED BY D. L. SCOTT

SYSTEMIC LUPUS ERYTHEMATOSUS, THROMBOCYTOPENIA, MICROANGIOPATHIC HAEMOLYTIC ANAEMIA AND ANTI-CD36 ANTIBODIES R. AL-SHAHI, J. C. MASON, R. RAO, C. HURD,† E. M. THOMPSON,* D. O. HASKARD and K. A. DAVIES Rheumatology Unit and *Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN and †Platelet Immunology Laboratory, East Anglian Blood Centre, Long Road, Cambridge CB2 2PT SUMMARY Thrombocytopenia in patients with acute systemic lupus erythematosus (SLE) frequently presents the clinician with considerable diagnostic and therapeutic difficulties. In this Grand Round, we present a 48-yr-old woman with a 7 yr history of lupus, who presented with acute proliferative glomerulonephritis and nephrotic syndrome, pneumonia, profound hypocomplementaemia and a severe microangiopathic haemolytic anaemia with associated thrombocytopenia. Her thrombocytopenia proved initially refractory to conventional immunosuppressive therapy, and corticosteroids, and resolved only with plasma exchange and repeated fresh frozen plasma infusions. Serological testing revealed high-titre antinuclear antibodies (ANA) and markedly raised antibodies to double-stranded (ds) DNA, but no significant elevation in anticardiolipin antibodies. Platelet-associated IgG and IgM and antibodies to the CD36 glycoprotein antigen, expressed on platelets and endothelium, were detected in the serum. We address some of the difficult diagnostic and management issues raised by this complex patient and the possible immunopathological links between antibodies to CD36, immune-mediated red cell destruction, thrombocytopenia and thrombotic microangiopathic haemolytic anaemia. K : Systemic lupus erythematosus, Thrombocytopenia, Thrombotic microangiopathic haemolytic anaemia, Anti-CD36.

112 × 109/l, white cell count 6.2 × 109/l, absolute reticulocyte count 118.6 × 109/l (normal range 25– 85 × 109/l). Antiglobulin test (DAGT) was negative and fibrin degradation products (FDPs) were 16– 32 mg/l. Haptoglobins were undetectable; haematinics and a coagulation screen were normal. Microscopic examination of the peripheral blood film revealed schistocytes (fragmented erythrocytes), occasional spherocytes, anisocytosis and poikilocytosis, fibrin strands and few platelets (Fig. 1). Immunoglobulins, urea and electrolytes were within normal limits, but albumin was 15 g/l (normal range 35–55 g/l), uric acid 0.71 mmol/l (normal range 0.1–0.4 mmol/l), aspartate aminotransferase (AST) 45 IU/l (normal range 5–35 IU/l) and lactate dehydrogenase (LDH) 1280 IU/l (normal range 120– 500 IU/l). The erythrocyte sedimentation rate (ESR) was 76 mm in the first hour and C-reactive protein (CRP) 89 mg/l (normal Q10 mg/l). Urinalysis revealed 5.6 g protein/24 h with an active sediment containing granular and red cell casts, and a creatinine clearance of 68 ml/min. Further immunological investigations later revealed profound hypocomplementaemia with a C3 of 0.22 g/l, C4 of 0.03 g/l and a CH50 Q 10% of the Normal Human Pool (NHP); double-stranded (ds) DNA binding was 91% (normal range Q30%) and antinuclear antibody titre was 1:10 240. Anticardiolipin IgG and IgM were within the normal range; VDRL and lupus anticoagulant were negative. Antibodies to Sm and RNP antigens were also detected, but antineutrophil cytoplasmic antibodies (ANCA) were negative. A plain chest X-ray showed right lower zone consolidation and cardiomegaly,

CASE REPORT A 48-yr-old Black woman, originally from Jamaica, was admitted to the Hammersmith Hospital with a 2 month history of facial rash, joint pains, hair loss, mouth ulcers and malaise. She also gave a 2 week history of breathlessness and a productive cough. Systemic lupus erythematosus (SLE) had been diagnosed 7 yr previously following presentation to her local hospital with an autoimmune haemolytic anaemia (AIHA). The subsequent course of her disease had been complicated by steroid-induced hypertension, azathioprine-induced leucopenia, oesophageal candidiasis, and a flare of her disease, affecting primarily her skin and large joints. Her past medical history also included hypothyroidism and an iron-deficiency anaemia secondary to menorrhagia. During the 6 months prior to her presentation, the patient had been poorly compliant with her treatment regimen, which comprised prednisolone EC 10 mg/15 mg on alternate days, azathioprine 100 mg daily (which she had completely omitted for 2 months), nifedipine, ranitidine and thyroxine. On examination at the time of admission, she was pyrexial (38°C), with a butterfly rash, facial post-inflammatory hyperpigmentation and oral ulceration. Blood pressure was 190/110 mmHg and the patient was noted to have muffled heart sounds and a laterally displaced apex beat. There was peripheral and peri-orbital oedema, and signs of right basal lung consolidation. Initial investigations revealed the following: haemoglobin 6.4 g/dl, MCV 96.4 fl, platelet count Accepted 20 January 1997. Correspondence to: K. A. Davies.

= 1997 British Society for Rheumatology

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subsequently confirmed by echocardiography to be due to a combination of left ventricular hypertrophy and a pericardial effusion. Renal ultrasound demonstrated increased parenchymal echogenicity and renal biopsy showed a proliferative glomerulonephritis (WHO grade III or IV), some mild tubular loss, a focal chronic interstitial infiltrate and hyaline changes of the blood vessels (Fig. 2). There was no evidence of thrombi nor fragmented red blood cells (RBCs) within the renal vascular lumina. The bone marrow trephine biopsy was mildly hypercellular with increased erythropoiesis and megakaryocyte numbers. The working diagnoses at this time were active SLE complicated by nephritis manifest as a nephrotic syndrome, a microangiopathic haemolytic anaemia (MAHA) and a right lower lobe pneumonia. Our initial management comprised prednisolone EC 60 mg daily and a 750 mg pulse of i.v. cyclophosphamide which was subsequently repeated on six occasions at 3–5 week intervals. Salt and fluid intake were restricted, and i.v. diuretic and human albumin infusions commenced. In addition to conventional antibiotics for a community-acquired pneumonia, she was given prophylactic oral penicillin, amphotericin lozenges and s.c. heparin. Four different antihypertensive drugs were required to treat refractory hypertension. During the acute and convalescent phases of the patient’s illness, serum was sent for immunohaematological investigations. Platelet-associated immunoglobulin was strongly positive for IgG and IgM by the direct platelet immunofluorescence test. An array of platelet glycoprotein-specific antibodies were screened for, by testing the serum by a solid-phase ELISA PakPlus kit (GTI, Brookfield, USA). The only serum antibodies detected to the platelet glycoproteins tested during the acute phase were directed against CD36 (glycoprotein IV) for which the ratio of test serum to negative control was 1.5. During the first 2 weeks of her admission, the patient remained persistently anaemic and developed

F. 1.—Blood film showing schistocytes, thrombocytopenia, anisocytosis, poikilocytosis and occasional spherocytes.

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F. 2.—Photomicrograph of the renal biopsy showing a glomerulus with proliferation and crescent formation. There is also rupture of Bowman’s capsule basement membrane. An obsolete glomerulus is seen (top right).

marked thrombocytopenia (platelet count at 10 days: 37 × 109/l) and peripheral blood films continued to show fragmented RBCs, fibrin strands, spherocytes and anisocytosis, compatible with a diagnosis of a MAHA. Four cycles of plasma exchange with fresh frozen plasma were attempted, but were complicated by line-related sepsis and gastrointestinal bleeding. Treatment with daily fresh frozen plasma (FFP) infusions, and supportive platelet and RBC transfusions, was subsequently administered for a further 2 weeks, with an improvement in platelet count and haemoglobin (Fig. 3). Further blood films showed significantly less red cell fragmentation. Serum creatinine and urea rose during the first 2 months of the patient’s admission, pari passu with diuretic and anti-hypertensive therapy, but renal function has subsequently returned to normal. Four weeks after admission, the patient was found on the ward unconscious with generalized twitching. Examination revealed she was hypertensive and pyrexial with a flaccid right-sided hemiparesis and hyper-reflexia. She sustained a full, but gradual recovery over 48 h. Examination of the cerebrospinal fluid (CSF) was essentially normal. Enhanced CT and MRI of the brain demonstrated deep white matter and periventricular small-vessel disease. An electroencephalogram (EEG) performed 1 day after the event showed diffuse slow-wave abnormalities with an absent alpha rhythm which had resolved 6 days later after treatment with an anticonvulsant, an antiviral agent and broad-spectrum antibiotics. The diagnosis was of a motor seizure with a prolonged post-ictal phase, secondary to a thrombotic microvasculopathy. The patient’s therapy was also complicated by recurrent Clostridium difficile enteritis. During the latter part of her 5 month illness, she developed a persistent, low-grade, grumbling pyrexia, diarrhoea

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and abdominal pain. Cryptosporidiosis was diagnosed by colonic biopsy and treatment with paromomycin led to the resolution of her fever and other symptoms. Five months after admission, the patient was discharged from hospital. Her SLE was serologically inactive at this time, and her nephritis had resolved with pulsed i.v. cyclophosphamide therapy. There has been no further evidence of opportunistic infection and no further seizures. Prophylactic phenytoin has been stopped. The platelet count is now normal, with no evidence of ongoing microangiopathy on the blood film. Convalescent anti-CD36 antibodies are comparable to a negative control. DISCUSSION In the discussion, we first address a number of diagnostic issues in this patient, and discuss her clinical management. We then consider the possible role of anti-CD36 antibodies in the immunopathogenesis of microangiopathy in the context of SLE. Diagnostic issues Our patient satisfied eight of the 11 ARA revised classification criteria for SLE [1]. Conventional laboratory markers indicated that her lupus was active at the time of presentation with her acute illness, namely a raised ESR, hypocomplementaemia, high-titre ANA and markedly raised antibodies to dsDNA. Notably, neither significantly elevated anticardiolipin antibodies nor a lupus anticoagulant

F. 3.—Platelet count and serum creatinine during the first 4 months of the patient’s illness.

were detected. The diagnosis of a MAHA was based on the characteristic blood film, anaemia, reticulocytosis, negative DAGT and ensuing thrombocytopenia. The modest elevation of FDPs implied low-grade fibrinolysis, but there was no other evidence of disseminated intravascular coagulation such as deranged coagulation or excessive fibrinogen consumption. A severe MAHA, in the context of active SLE and thrombocytopenia, posed both diagnostic and therapeutic dilemmas. Plasma exchange proved technically difficult, in view of the patient’s initial severe thrombocytopenia, and was complicated by recurrent life-threatening gastrointestinal haemorrhage. The cause of the sudden altered level of consciousness, hemiplegia and presumed seizure, 4 weeks into the patient’s illness, remains unclear. MRI confirmed widespread small-vessel disease and her neurological deterioration was attributed at the time to a non-inflammatory thrombotic microvasculopathy. EEG findings did not support a diagnosis of herpes simplex encephalitis, and CSF examination was not diagnostic of acute inflammatory cerebral lupus, revealing only a marginally elevated protein level, and no leucocytes. A thrombotic microangiopathy affecting the CNS is well described in the antiphospholipid syndrome (APLS) (primary, or in the context of SLE), but our patient did not have elevated levels of antiphospholipid antibodies. Antibodies to the glycoprotein CD36, an autoantibody specifically associated with ‘thrombotic microangiopathic haemolytic anaemia’ (TMHA), were detected in the patient’s serum. The term TMHA was first introduced [2] to describe conditions in which localized or diffuse microvascular thrombosis occurs. TMHA encompasses a spectrum of disorders including thrombotic thrombocytopenic purpura (TTP), haemolytic–uraemic syndrome (HUS), malignant hypertension, post-partum renal failure, pre-eclampsia and scleroderma renal crisis. The typical clinical picture may be complicated by thrombocytopenia, MAHA, fever, neurological symptoms and/or renal dysfunction, but need not have all these manifestations. Previously, this has led many authors to invoke the diagnosis of TTP to describe such phenomena occurring in patients with SLE, especially when symptoms such as headache, seizures, altered states of consciousness and strokes predominate. An autopsy study has shown that TTP-like illnesses may be underdiagnosed ante mortem in SLE [3]. There have been a number of recent reports of TMHA complicating lupus [4, 5]. Nesher and colleagues [4], in a review of 28 such patients, all of whom were female, found 50% to have clinically and serologically active SLE at presentation (all with hypocomplementaemia). Twenty-five patients presented with a TTP-like clinical picture and lupus anticoagulant or anti-cardiolipin antibodies were positive in five cases. Haemolytic– uraemic syndrome was the presentation in three patients out of the 28. These clinical and laboratory features in this study were similar to those in a series of seven patients described by Jain and co-workers [5],

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in which the majority of patients had elevated LDH, low haptoglobin levels (in those tested) and a negative direct antiglobulin test. Implications for treatment Of those patients with co-existent SLE and TMHA who have been reported in the literature, the most commonly employed treatments were plasmapheresis or plasma infusion, and high-dose corticosteroids [4, 5]. In the study of Nesher and colleagues [4], a 25% mortality was observed in patients treated with plasma infusion or plasmapheresis, compared with 57% in those treated with steroids and immunosuppression alone. The patient described in the present report showed little response to initial immunosuppressive therapy, and her thrombocytopenia only resolved after four cycles of plasma exchange and subsequent daily FFP infusions, over a period of 3 weeks (Fig. 3). The efficacy of plasmapheresis in this condition has been taken to imply that a humoral immune mechanism is involved, and we discuss below some of the possible immunopathological mechanisms which may be implicated. Immunopathological mechanisms The causes of thrombocytopenia in SLE can be divided into three main categories: failure of production (due to drug treatment or diseaseassociated myelosuppression), abnormal distribution (such as pooling in the spleen), or excessive destruction (as may occur in APLS, MHA or direct antibody-mediated thrombocytopenia). The aetiology of TMHA is poorly understood, but it is likely that platelets, humoral factors (antibody and complement), and the microvascular endothelium all play an important role in pathogenesis. Small-vessel injury triggers platelet adhesion to the endothelium and aggregation, with concomitant diminution of PGI2 and increased synthesis of thromboxanes. Local binding of antibody or immune complexes from the circulation may also promote microthrombus formation, local complement activation, and further endothelial damage. In addition to the wellcharacterized autoantibodies to antigens such as DNA, RNP and C1q in lupus, specific antibodies to a number of target antigens on platelets, RBCs and endothelium have been described. Circulating antiplatelet antibodies have been reported in patients with TTP and SLE [6]. The TMHA syndrome has been most frequently described in patients with active lupus, in whom ongoing tissue damage and complement activation are occurring. In this situation, the presentation of neoepitopes on cellular or fluid-phase antigens may occur, with the resultant stimulation of further autoantibody production, initiating an inflammatory vicious cycle (Fig. 4). Negatively charged phospholipids, which may be exposed subsequent to cell membrane disruption, are clearly important potential targets for autoimmunity in this context, e.g. in APLS. However, there is also

F. 4.—Suggested schema for the immunopathology of TMHA (see Discussion).

increasing interest in antibodies to a recently described molecule: glycoprotein CD36. Autoimmunity to this antigen has been implicated in the pathogenesis of microvascular thrombosis in TTP [7] and in patients with lupus anticoagulant [8]. It is an 88 kDa leucocyte differentiation antigen [9] which is immunologically related to platelet glycoprotein IV (GPIV). CD36 is expressed on the membranes of platelets (and monocytes), mediating signal transduction and cellular activation by interactions with a range of ligands, including the extracellular matrix proteins thrombospondin and collagen [10–13]. CD36 is expressed in low levels on the surface of RBCs in normal human blood [14]. Its preferential expression on microvascular endothelium has been demonstrated both in vivo and in vitro. Human dermal microvascular endothelial cells express cell surface CD36, whereas large-vessel endothelial cells derived from umbilical veins do not [15]. It is, therefore, possible that anti-CD36 antibodies may contribute to vascular injury and TMHA by reacting with the antigen on both platelets and small-vessel endothelium, causing activation, upregulation of adhesion molecule expression, and the local production of chemokines and other inflammatory mediators. Thrombocytopenia may result from increased immune-mediated platelet clearance, or the local formation of microthrombi on the subendothelium as a consequence of endothelial cell activation and detachment. CD36 may be an obligate receptor for platelet activation in some cases of TTP [7] and it has also been suggested that CD36-dependent activation might involve the binding to the anti-CD36 antibodies of the platelet agglutinating protein p37 [16]. CD36 also has a role in mediating neutrophil apoptosis [17], and as a ‘scavenger’ receptor for oxidized low-density lipoprotein [18]. Autoantibodies to CD36 which impair the function of this receptor might, therefore, inhibit the clearance of apoptotic neutrophils by monocytes and macrophages, with pro-inflammatory consequences. Similarly, an inhibitory autoantibody might impair the binding of oxidized low-density lipoprotein, a molecule which is now regarded as of vital importance in the initiation and progression of thrombosis, atherogenesis and inflammation.

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Concluding remarks In summary, we have described a patient with active SLE with hypocomplementaemia and TMHA. Therapy with high-dose corticosteroids and immunosuppression was only partly effective, and the thrombotic microangiopathy only resolved with adjunctive treatment with plasma exchange and FFP infusion. Anti-CD36 antibodies were detected in the patient’s serum at the height of the illness, but not in convalescent serum. We have discussed the possible immunopathological mechanisms underlying the development of microangiopathy in lupus, and the potential role of anti-CD36 antibodies. We suggest that the measurement of this recently described autoantibody may be useful in the management of patients with severe thrombocytopenia occurring in the context of SLE. A We are grateful to Dr Willem H. Ouwehand, University Lecturer in Transfusion Medicine at the University of Cambridge, for his assistance with the platelet immunology. R 1. Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. 2. Symmers W. Thrombotic microangiopathic haemolytic anaemia (thrombotic microangiopathy). Br Med J 1952;2:897–903. 3. Devinski O, Petito CK, Alonso DR. Clinical and neuropathological findings in systemic lupus erythematosus: the role of vasculitis, heart emboli and thrombotic thrombocytopenic purpura. Ann Neurol 1988;23:380–4. 4. Nesher G, Hanna VE, Moore TL, Hersh M, Osborn TG. Thrombotic microangiopathic hemolytic anemia in systemic lupus erythematosus. Semin Arthritis Rheum 1994;24:165–72. 5. Jain R, Chartash E, Susin M, Furie R. Systemic lupus erythematosus complicated by thrombotic microangiopathy. Semin Arthritis Rheum 1994;24:173–82. 6. Itoh Y, Sekine H, Hosono O et al. Thrombotic thrombocytopenic purpura in two patients with systemic

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lupus erythematosus: clinical significance of anti-platelet antibodies. Clin Immunol Immunopathol 1990;57:125– 36. Tandon NN, Rock G, Jamieson GA. Anti-CD36 antibodies in thrombotic thrombocytopenic purpura. Br J Haematol 1994;88:816–25. Rock G, Chauhan K, Jamieson GA, Tandon NN. Anti-CD36 antibodies in patients with lupus anticoagulant and thrombotic complications. Br J Haematol 1994;88:878–80. Tandon NN, Lipsky RH, Burgess WH, Jamieson GA. Isolation and characterization of platelet glycoprotein IV (CD36). J Biol Chem 1989;264:7570–5. Aiken ML, Ginsberg MH, Byers WV, Plow EF. Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression. Blood 1990;76:2501–9. Clemetson KJ. Platelet activation: signal transduction via membrane receptors. Thromb Haemostasis 1995;74: 111–6. Tandon NN, Kralisz U, Jamieson GA. Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion. J Biol Chem 1989;264:7576– 83. Diaz-Ricart M, Tandon NN, Carretero M, Ordinas A, Bastida E, Jamieson GA. Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in flowing whole blood. Blood 1993;82:491–6. van Schravendijk MR, Handunnetti SM, Barnwell JW, Howard RJ. Normal human erythrocytes express CD36, an adhesion molecule of monocytes, platelets, and endothelial cells. Blood 1992;80:2105–14. Swerlick RA, Lee KH, Wick TM, Lawley TJ. Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. J Immunol 1992;148:78–83. Siddiqui FA, Lian EC. Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36). Biochem Int 1992;27:485–96. Savill J, Hogg N, Ren Y, Haslett C. Thrombospondin co-operates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 1992;90:1513–22. Rigotti A, Acton SL, Krieger M. The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids. J Biol Chem 1995;270:16221–4.