ARTHRITIS & RHEUMATISMVol. 52, No. 1, January 2005, pp 262–268DOI 10.1002/art.20718© 2005, American College of Rheumatology

Induction of Remission by B Lymphocyte Depletionin Eleven Patients With Refractory

Antineutrophil Cytoplasmic Antibody–Associated Vasculitis

Karina A. Keogh,1 Mark E. Wylam,1 John H. Stone,2 and Ulrich Specks1

Objective. To assess the clinical effects of ritux-imab therapy in patients with antineutrophil cyto-plasmic antibody (ANCA)–associated vasculitis (AAV).

Methods. The study group comprised 11 patientswho had active AAV despite receiving maximally toler-ated doses of cyclophosphamide or had contraindica-tions for cyclophosphamide use. All patients had ANCAreactive against proteinase 3. The patients receivedrituximab infusions and glucocorticoids to induce re-mission. Three patients also received plasma exchange.No other immunosuppressive agents were used. Glu-cocorticoids were tapered as soon as control of diseaseactivity was achieved. Disease activity was monitoredusing the Birmingham Vasculitis Activity Score, modi-fied for Wegener’s granulomatosis.

Results. Rituximab therapy was well tolerated byall patients, and adverse events were rare. Following therituximab infusions, circulating B lymphocytes becameundetectable, and ANCA titers decreased significantly.Remission was achieved in all patients and was main-tained while B lymphocytes were absent.

Conclusion. The ability to achieve stable remis-sions with rituximab in patients with AAV refractory toconventional therapy suggests that B lymphocyte deple-tion may be a safe, effective, mechanism-based treat-ment modality for treatment of patients with theseconditions.

Wegener’s granulomatosis (WG) and micro-scopic polyangiitis are potentially fatal forms of systemicvasculitis associated with antineutrophil cytoplasmicantibodies (ANCA). Cyclophosphamide remains themost effective treatment for ANCA-associated vasculitis(AAV) with severe organ- or life-threatening diseasepresentations (1). Selective suppression of immunoglob-ulin secretion by human B lymphocytes is thought to bean important mechanism explaining the efficacy of cyclo-phosphamide in AAV (2). Unfortunately, in �10% ofpatients the disease remains refractory to conventionaltherapy, and these patients experience severe, dose-limiting side effects of cyclophosphamide (1). Few treat-ment options are available for such patients.

Rituximab, a chimeric monoclonal antibody, in-duces selective depletion of B lymphocytes by targetingthe CD20 surface antigen. Rituximab is an establishedtreatment of non-Hodgkin’s lymphoma, and interest inits use for autoimmune disease is growing (3). Antici-pating that B lymphocyte depletion with rituximab, anda potential downstream effect on plasma cells, wouldresult in the resolution of vasculitic symptoms anddisappearance of ANCA and allow the tapering ofglucocorticoids without relapse, we used rituximab totreat 11 patients with severe AAV refractory to conven-tional therapy, on a compassionate-use basis.

PATIENTS AND METHODS

The study group comprised 11 consecutive patientswho received treatment for severe AAV refractory to conven-tional therapy between January 2000 and September 2002. Thereview of these cases was approved by the Institutional ReviewBoard. Patients were defined as having “refractory disease” ifthey had active disease that had not been controlled by themaximally tolerated cyclophosphamide dose given in conjunc-tion with glucocorticoids (n � 8) or if treatment with cyclo-phosphamide was contraindicated because of previous drug-induced cystitis (n � 1) or profound and prolonged cytopenias(n � 2).

Drs. Keogh, Wylam, and Specks’ work was supported by theMayo Foundation. Dr. Stone’s work was supported in part by the NIH(grant K24-AR-049185-01).

1Karina A. Keogh, MB, BCh, Mark E. Wylam, MD, UlrichSpecks, MD: Mayo Clinic, Rochester, Minnesota; 2John H. Stone,MD, MPH: Johns Hopkins University, Baltimore, Maryland.

Address correspondence and reprint requests to UlrichSpecks, MD, Division of Pulmonary and Critical Care Medicine, MayoClinic and Foundation, 200 First Street SW, Rochester, MN 55905.E-mail: specks.ulrich@mayo.edu.

Submitted for publication January 14, 2004; accepted inrevised form September 14, 2004.

262

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B LYMPHOCYTE DEPLETION FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 263

Clinical activity was assessed using a disease-specificactivity index, the Birmingham Vasculitis Activity Score mod-ification for WG (BVAS/WG) (4). BVAS/WG scores weredetermined by consensus between 2 physician-investigators(KK and US). “Severe disease” was defined as disease thatposed an immediate threat to the patient’s life or the functionof a critical individual organ (items classified as “major” on theBVAS/WG instrument [4] and generally deemed to requiretherapy with cyclophosphamide). Remission was defined as aBVAS/WG score of 0 (4).

The clinical characteristics of the patients are summa-rized in Table 1. The median age of the patients (6 men and 5women) was 31 years (range 14–71 years). The mean (�SD)disease duration was 57.8 � 48.5 months. Ten patients hadWG, and 1 had microscopic polyangiitis. All patients hadactive, severe disease, with BVAS/WG scores ranging from 3 to11. Four patients had alveolar hemorrhage, which was docu-mented by bronchoscopy with bronchoalveolar lavage in 3.(The fourth patient had hemoptysis, a decline in the hemoglo-bin concentration, and the rapid onset of ground-glass infil-trates on computed tomography of the chest in the absence ofinfection.) Five patients had active renal disease, which wasdocumented by red blood cell (RBC) casts on urine micros-copy; 3 of these patients had active glomerulonephritis con-firmed by renal biopsy.

Glomerular filtration rates were estimated using theCockroft-Gault equation. B cells were measured byfluorescence-activated cell sorting (FACS) analysis usingCD19 as marker, with a lower detection limit of 1 cell permicroliter. ANCA were determined at the Mayo Clinic Roch-ester Clinical Immunology laboratory using previously de-scribed procedures (5). The current procedure for ANCAtesting, which was used in most of the patients in this study, isto perform proteinase 3 ANCA (PR3 ANCA)–specific directenzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence assays together. Before implementation of thispractice early in the course of this study, patients for whomclassic ANCA (cANCA) immunofluorescence testing had beenperformed had PR3 ANCA reactivity confirmed by captureELISA (5). In our laboratory, cANCA titer determinationshave an interobserver and intraobserver variability of �1dilution. Each titer is determined by 2 independent readers,and in case of disagreement, by a third reader. The average isreported as the final titer. The laboratory personnel wereblinded to patients’ clinical diagnoses or status.

The treatment protocol included rituximab (4 weeklydoses of 375 mg/m2) and prednisone (up to 1 mg/kg/day),which was tapered once disease activity improved. In 8 pa-tients, prednisone was preceded by intravenous methylpred-nisolone (1 gm/day for 3 days). Three patients with renaldisease also received plasma exchange before receiving ritux-imab: patient 2 had three 3-liter replacements with fresh frozenplasma, patient 5 had six 4.5-liter replacements with albuminsolution, and patient 9 had three 5-liter replacements with 1:4fresh frozen plasma and albumin.

Data collection was censored on June 30, 2003. Theinitial treatment outcome of the index case at 18 months hasbeen reported previously (6). Comparisons were performedusing JMP software, version 5.0 (SAS Institute, Cary, NC).Student’s paired t-tests were used when similar measurementswere obtained in the same patient at different time intervals.

For nonparametric data (BVAS/WG, ANCA), comparisonswere made using a Wilcoxon’s/Kruksal-Wallis test. Data arepresented as the mean � SD or median with interquartileranges. P values less than 0.05 were considered significant.

RESULTS

Following rituximab therapy, peripheral blood Blymphocytes became undetectable in all patients. Dis-ease remission was induced in all patients (Figure 1A),and glucocorticoids were tapered successfully in all(median dose 0 mg [range 0–1.5 mg]). Ten patientsachieved BVAS/WG scores of 0 within 6 months. Onepatient (patient 10) had persistent lung nodules but noevidence of active disease in other organs. These nod-ules were given a score of 1 point (persistent BVAS/WGitem) for as long as they were shrinking (4 months).Once no further change occurred (8 months), they wereconsidered to be damage rather than active disease, andthe BVAS/WG score was 0.

Six months after the initiation of rituximab ther-apy, significant reductions in the erythrocyte sedimenta-tion rate (ESR) and C-reactive protein (CRP) level wereobserved. The mean (�SD) ESR fell from 48.9 � 48mm/hour to 19 � 22 mm/hour (n � 7; P � 0.02), and themean (�SD) CRP level fell from 9.1 � 9.5 mg/dl to1.1 � 1 mg/dl (n � 5; P � 0.002).

All patients were cANCA/PR3 ANCA positivebefore and at the time of rituximab infusion, with a mean�SD of 4.1 � 3 measurements preceding the firstinfusion. The baseline measurements shown in Figure1B were obtained 5.3 � 7 days before the start ofinfusion. Titers of cANCA decreased significantly in allpatients, with 8 of the 11 patients becoming ANCAnegative (Figure 1B).

For the 5 patients with active renal disease, thecalculated mean � SD creatinine clearance was 34 � 25ml/minute at baseline and 46 � 33 ml/minute at 6months (P � 0.48). Serial serum creatinine determina-tions for the individual patients are shown in Figure 1D.Two patients who were dialysis dependent before theinitiation of rituximab also received plasma exchange.Both were able to discontinue dialysis. In another pa-tient with renal involvement, the serum creatinine levelincreased from 1.3 gm/dl to 2.8 gm/dl within the first 3weeks of therapy but then stabilized. Renal function inthe other 2 patients with active renal disease remainedunchanged during the period of observation. The micro-scopic hematuria with RBC casts resolved by 6 monthsin all 5 patients, and protein concentrations of spot urine

264 KEOGH ET AL

samples fell from a mean � SD of 120 � 66 mg/dl to56.8 � 50 mg/dl (P � 0.008).

Following completion of glucocorticoid tapering,remission was maintained in every patient as long as Blymphocytes were undetectable. B lymphocytes returned

in 9 patients during the followup period (4–12 monthsafter infusion). Three of these 9 patients remainedANCA negative and experienced disease in remission (5,8, and 10 months after return of B lymphocytes). Two ofthe 9 patients experienced a relapse after discontinua-

Figure 1. Clinical outcomes of patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis. A, Graphshowing rapid decline in scores on the Birmingham Vasculitis Activity Score index modified for Wegener’s granulomatosis(BVAS/WG) following initiation of rituximab therapy. Arrows indicate re-treatment with rituximab and glucocorticoids in the2 patients with disease relapse (patients 3 and 4). As of June 30, 2003 (mean followup 16 months, range 10–42 months), diseasewas in remission in all patients. B, Graph showing decline in ANCA levels after initial treatment with rituximab. Patients 1, 7,and 10 had asymptomatic ANCA titer increases. Patients 3 and 4 had elevations in ANCA titers in association with diseaseflares. Patient 8 received a cadaveric kidney transplant at 5 months and underwent no further ANCA testing. The mean � SDtime to an increase in ANCA titers, when it occurred, was 6.8 � 3 months. Time to re-treatment ranged from 1 day to 3 weeksfollowing ANCA rise or disease flare. C, Graph showing serial ANCA titers following re-treatment with rituximab. Patient 1received no glucocorticoids with either his first or second rituximab re-treatment. Patient 3 was re-treated for a flare of diseaseand also received intravenous methylprednisolone. Patient 4 was re-treated for a flare of disease and received prednisone 40mg daily. At the time of re-treatment for asymptomatic rises in ANCA titers, patients 7 and 10 were still receiving prednisone,2.5 mg and 8 mg daily, respectively. Their dosages were not changed. Data for patient 9, who was also re-treated preemptivelyfor an asymptomatic ANCA titer increase (and who was receiving prednisone 40 mg daily for 1 week as infusionpremedication), are not shown, because no followup ANCA titer was available. D, Graph showing that average renal functionremained stable in the 5 patients with active renal vasculitis. In A, B, and D, data are also presented as box plots, where the boxesrepresent the interquartile range, the bar within the boxes represents the median, and the bars outside the boxes represent therange outside the quartiles. � � P � 0.0001.

B LYMPHOCYTE DEPLETION FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 265

tion of glucocorticoid therapy (at 7 and 12 monthsfollowing rituximab treatment, respectively). Both re-lapses were associated with normal B lymphocyte countsand elevations in ANCA titers. Remissions were re-stored in both patients by prompt reinstitution of therituximab/glucocorticoid regimen. Three of the 9 pa-tients whose B lymphocytes recovered after treatmentwere re-treated preemptively with rituximab withoutglucocorticoids for rising ANCA titers (Figure 1C). Inall 3 patients, disease remained in remission duringfollowup. Finally, 1 patient who never became ANCAnegative (albeit her baseline titers declined markedly)was re-treated preemptively when her ANCA titer roseand had no disease flare during the observation period.

Infusion-related adverse events were rare. Onepatient experienced mild angioedema following the firstinfusion, which was followed by polyarthritis after thefourth infusion. The polyarthritis was attributed to se-rum sickness and responded to short-term glucocorti-coid treatment. Another patient described dizziness withthe first infusion but tolerated subsequent infusions well.Other adverse events are included in Table 2, along withcomments regarding their potential attribution. All 7patients who had immunoglobulin determinations aftertheir rituximab infusions had reduced IgM levels(mean � SD nadir 14.3 � 13.5 mg/dl; normal 60–300mg/dl) and mildly reduced IgG levels (mean � SD nadir429.3 � 145 mg/dl; normal 600–1,500 mg/dl).

DISCUSSION

The only proven therapy for severe AAV—cyclo-phosphamide combined with glucocorticoids—is associ-ated with substantial morbidity and many potentially

dose-limiting adverse effects (7,8). Many of the deathsthat occur during the treatment of AAV are related totherapy (1,7,8). Furthermore, a subset of patients fail toachieve stable remissions even with the maximally toler-ated doses of cyclophosphamide (7,8). Consequently,safe and effective alternatives to cyclophosphamide areneeded for the treatment of AAV. This report ofsuccessful remission induction using rituximab in 11patients with disease refractory to conventional treat-ment has several important implications.

First, the fact that in all patients disease re-mained in remission during the period of B lymphocytedepletion supports a central pathogenic role for Blymphocytes in AAV (2,9). B lymphocytes may exerttheir pathogenic effects in AAV via several mechanisms,including costimulation, antigen presentation, and auto-antibody production.

Second, the observation that disease remained inremission after restoration of B lymphocyte counts for aslong as ANCA remained undetectable provides addi-tional evidence for the importance of ANCA in diseasepathophysiology. Prospective clinical studies have shownthat the persistence or recurrence of ANCA in patientswho have achieved clinical remission is a predictor ofrelapse (10). Many in vitro studies designed to investi-gate the pathogenic role of ANCA have identified avariety of proinflammatory effects of ANCA, and adirect pathogenic role of ANCA in vivo has now beendocumented in mice (11). Consequently, therapeuticinterventions aimed at elimination of ANCA or atsuppression of ANCA production are appealing. Com-pared with cyclophosphamide, rituximab may contributeto this goal in a more selective manner.

Table 2. Adverse events in patients with antineutrophil cytoplasmic antibody–associated vasculitis*

Patient Adverse event Comments

1 Presumed bacterial bronchitis (2 episodes) Prompt response to oral antibiotics2 None3 Viral upper respiratory tract infection with conjunctivitis Spontaneous resolution4 Lower extremity petechiae (days 3–6 postinfusion) Spontaneous resolution5 Dizziness (during first infusion); MRSA sepsis with

vertebral osteomyelitis following dialysis catheterplacement

MRSA olecranon bursitis prior to rituximab treatment

6 Multiple exacerbations of respiratory infection Known bronchiectasis; no change in frequency of infectionsbefore or after therapy

7 Thrombocytopenia (platelet count 43,000/mm3) Timing and duration suggest that the cause was dapsone8 Hypertension (during third infusion) Volume-sensitive hemodialysis patient with similar prior episodes9 None

10 None11 Mild angioedema (during first infusion); polyarthritis

following fourth infusionResponded to a short burst in steroid dose

* MRSA � methicillin-resistant Staphylococcus aureus.

266 KEOGH ET AL

Third, our results suggest that either B lympho-cytes or short-lived plasma cells (or both) rather thanlong-lived plasma cells are the cellular source of ANCA.ANCA titers declined in all of our patients and becamenegative in most. Long-lived plasma cells are not af-fected by anti-CD20 therapy. Consequently, as in otherautoimmune diseases (12), B lymphocytes and short-lived plasma cells (which are dependent on antigen-specific B lymphocyte progenitors) seem to be primarilyresponsible for autoantibody production. However, ourpatients who had the highest initial ANCA titers did notbecome ANCA negative and subsequently had rises inthe ANCA titer when B lymphocytes returned. Thus, forreasons that remain to be investigated formally, a singlecourse of rituximab may not eliminate all ANCA-producing cells or their precursors in some patients.

The fact that in 3 patients ANCA did not returnafter reconstitution of B lymphocytes supports thehypothesis that rituximab therapy may result in restora-tion of tolerance to ANCA target antigens in AAV. Ifanti-CD20 therapy effectively eliminates B lymphocytes,there is a chance that the entire B cell lineage is restoredonce B lymphocyte production resumes. Antigens pre-sented to immature lymphocytes tend to trigger toler-ance by inducing anergy or elimination (13). In contrast,when antigen is presented only after the cells havematured and migrated to the periphery, cellular activa-tion is the typical result (13). Although conventionalimmunosuppression failed to suppress ANCA produc-tion in our 11 patients, it does induce long-term ANCAnegativity in some patients. The ability of rituximab torestore immune tolerance requires formal study.

Our study has several limitations, most of whichare related to the fact that it was not a prospectiveclinical trial. The potentially confounding effects ofconcomitant therapies require consideration. The use ofhigh-dose glucocorticoids in all patients undoubtedlyfacilitated the rapid induction of remission. However,most patients had active disease despite receiving signif-icant doses of oral glucocorticoids and other immuno-suppressants before receiving rituximab infusions. B celldepletion, in contrast, enabled induction and mainte-nance of remission despite subsequent tapering of glu-cocorticoids. This goal had not been achieved in thesepatients with any other regimen previously used. High-dose glucocorticoid therapy may also have contributedto the more precipitous fall in ANCA titers observedafter initial treatment compared with that observed inthe 3 patients who received rituximab alone preemp-tively. Other possible explanations for this observationinclude differences in sampling frequency and the fact

that measured ANCA titers are the result of the balancebetween ANCA production and consumption. At thetime of active systemic inflammation, ANCA consump-tion may result from ANCA binding to target antigenexpressed on the surface of activated neutrophils andmonocytes or released from these cells. In contrast,during remission, only the inhibition of ANCA produc-tion by rituximab may be a factor, and the rate of ANCAdecline is a reflection of the normal half-life of IgG.

Plasma exchange may also have contributed toremission induction in the 3 patients who received thistherapy concomitantly. It is remarkable, however, thatnone of the patients experienced disease flare followingglucocorticoid withdrawal. The overall responses of the5 patients with active renal disease to this rituximab/glucocorticoid regimen is very encouraging, particularlyin view of the inability of other treatment approaches tocontrol the disease safely. Further prospective studies ofrituximab in patients with renal vasculitis associated withANCA are indicated.

The lack of frequent B lymphocyte and ANCAdeterminations at predefined intervals in all patientsdoes not allow us to state with certainty that therecurrences of ANCA or increases in ANCA titers werealways preceded by the recurrence of B lymphocytes.However, the recurrence of ANCA or increases inANCA titers were not detected in the absence ofreconstituted B lymphocyte counts, and B lymphocytereconstitution preceding the recurrence of ANCA orelevations of ANCA titers was documented in 3 cases.Furthermore, although FACS analysis of peripheralblood B lymphocytes represents the only practical way tomeasure these cells in humans, their numbers mayinadequately reflect the presence of pathogenically sig-nificant tissue B lymphocytes.

Finally, the occurrence of human antichimericantibody was not measured in our patients, because theassay is available only from the manufacturer of ritux-imab and because, to date, no evidence has been pub-lished suggesting that human antichimeric antibody for-mation is an important clinical phenomenon in patientstreated with the doses of rituximab that we used. Incontrast to what is observed with the use of otherchimeric monoclonal antibodies (e.g., infliximab), themechanism of action of rituximab itself might be ex-pected to prevent formation of human antichimericantibody. Nevertheless, this issue deserves further study.

Two smaller series of patients with AAV refrac-tory to conventional treatment who appeared to benefitfrom rituximab were described recently (14,15). In bothseries, rituximab was used in conjunction with other

B LYMPHOCYTE DEPLETION FOR REFRACTORY ANCA-ASSOCIATED VASCULITIS 267

immunosuppressive agents, including intravenous cyclo-phosphamide, mycophenolate mofetil, azathioprine, orleflunomide. In contrast, in our patients all immunosup-pressive agents other than glucocorticoids were discon-tinued before the institution of rituximab therapy.

In summary, our ability to achieve stable remis-sions using rituximab in patients with AAV refractory toconventional therapy suggests that B lymphocyte deple-tion may be a safe, effective, mechanism-based treat-ment modality for AAV. A randomized, double-blindedtrial of this medication is now under way.

REFERENCES

1. Jayne D, Rasmussen N, Andrassy K, Bacon P, Tervaert JW,Dadoniene J, et al. A randomized trial of maintenance therapy forvasculitis associated with antineutrophil cytoplasmic autoantibod-ies. N Engl J Med 2003;349:36–44.

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