Presented in part at the 33rd Annual Meeting of the Japanese Society for Apheresis held November 9–12, 2012 in Nagasaki, Japan.
Granulocyte and Monocyte Adsorption Apheresis for Refractory Skin Diseases due to Activated Neutrophils, Psoriasis, and Associated Arthropathy
Article first published online: 7 OCT 2013
© 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis
Therapeutic Apheresis and Dialysis
Special Issue: Contributions from the 33rd Annual Meeting of the Japanese Society for Apheresis. Guest Editor: Hidenori Matsuo
Volume 17, Issue 5, pages 477–483, October 2013
How to Cite
Sakanoue, M., Takeda, K., Kawai, K. and Kanekura, T. (2013), Granulocyte and Monocyte Adsorption Apheresis for Refractory Skin Diseases due to Activated Neutrophils, Psoriasis, and Associated Arthropathy. Therapeutic Apheresis and Dialysis, 17: 477–483. doi: 10.1111/1744-9987.12113
- Issue published online: 7 OCT 2013
- Article first published online: 7 OCT 2013
- Manuscript Received: FEB 2013
- Granulocyte and monocyte adsorption apheresis;
- Multi-centered study;
- Neutrophilic dermatosis
Granulocyte and monocyte adsorption apheresis (GMA), an extracorporeal apheresis instrument whose column contains cellulose acetate (CA) beads, is designed to remove activated granulocytes and monocytes. We previously demonstrated that GMA was useful for treating neutrophilic dermatoses and associated arthropathy as it adsorbs Mac-1 (CD11b/CD18)-expressing neutrophils to the CA beads by the binding of complement component (iC3b) and CD11b expressed on activated neutrophils. The objective of this study is to further assess the clinical effectiveness of GMA in the treatment of neutrophilic dermatoses and associated arthropathy. The effect of GMA for skin lesions and joint lesions was assessed in 44 and 23 patients, respectively. Mac-1 expression on peripheral neutrophils was measured by flow cytometry. Skin lesions and arthropathy improved in 39 of 44 patients (88.6%) and 22 of 23 (95.6%), respectively. Mac-1 (CD11b/CD18) expression on the peripheral neutrophils, 27.1 ± 6.66 MFI (mean fluorescence intensity) before treatment, was reduced to 17.9 ± 3.02 MFI by GMA (P < 0.05). Clinical effectiveness of GMA for the treatment of intractable neutrophilic dermatoses and associated arthropathy was further confirmed.
Granulocyte and monocyte adsorption apheresis (GMA) is an extracorporeal apheresis instrument designed to remove pathogenic granulocytes. It features a column containing cellulose acetate (CA) beads [1, 2]. Our previous clinical studies on the efficacy of GMA for treating various skin diseases and related arthropathy attributable to activated granulocytes yielded excellent results in patients with pyoderma gangrenosum (PG), pustular psoriasis (PP), psoriatic arthritis (PsA), Behçet's disease (BD), adult Still's disease, leg ulcers associated with rheumatoid arthritis, cutaneous allergic vasculitis (CAV), Reiter's disease (RD), and systemic lupus erythematosus (SLE) skin rash [3-12].
Activated granulocytes express Mac-1, a cell-surface adhesive molecule that belongs to the integrin family . CD11b (integrin αM subunit) combines with CD18 (integrin β2 subunit) to form Mac-1 [14, 15], known as complement receptor 3 (CR3) [16, 17]. The CA beads in the GMA column activate and adsorb complement component iC3b, a ligand for Mac-1 [2, 18]. Based on these facts, we previously demonstrated that the column traps activated granulocytes by binding Mac-1 expressed on the granulocytes to iC3b on the beads .
In the present study, we assessed the efficacy of GMA in 44 patients with various skin diseases and 23 patients with arthropathy attributable to activated granulocytes.
Patients and Methods
We enrolled 44 patients in this study. Of these patients, 18 had PP (four generalized and 14 palmoplantar form), five had PsA, one had psoriasis vulgaris (PV), nine had BD, and six had PG. The other five patients had RD, acrodermatitis continua of Hallopeau, CAV, Sweet's syndrome, and erythema nodosum (EN). Arthropathy was associated in 23 patients. Of 23 patients with arthropathy, 11 had PP (one generalized and 10 palmoplantar form), five had PsA, and four had BD. The other three patients had RD, CAV, and Sweet's syndrome (Table 1).
|Patient no.||Age/Sex||Diagnosis||Site of skin lesions||Affected joints||Concomitant medications and their duration prior to GMA|
|1||59/F||Pustular psoriasis||Face, trunk, arms, legs||Rt. shoulder joint, sternoclavicular joint, waist, rt. wrist joints, knee joints, toe joints||Methotrexate (2 mg) 2–1 cap/day 4 days, loxoprofen (60) 3 tab/day 1 week|
|2||39/M||Pustular psoriasis||Trunk, arms, legs||–||nb- UVB, PUVA, CyA 150–100 mg/day, 9 months|
|3||51/F||Pustular psoriasis||Arms, legs, buttocks||–||Prednisolone pulse, prednisolone p.o, Etretinate, PUVA-bath, DDS, Colchicine|
|4||37/F||Pustular psoriasis||Trunk, arms, legs||–||None|
|5||35/M||Palmoplantar pustulosis + Arthropathy||Palms, soles||Shoulder joints, sternoclavicular joint, waist, rt. Ilium||Acetaminophen (60 mg) 1tab, diclofenac sodium (25 mg) 1tab, single use|
|6||77/F||Palmoplantar pustulosis||Arms, legs, palms, soles||–||None|
|7||38/F||Palmoplantar pustulosis + Arthropathy||Palms, soles||Sternoclavicular joint, hip joint, finger joint||Loxsoprofen (60 mg) 1 tab, Single Use|
|8||57/F||Palmoplantar pustulosis||Palms, soles||–||PUVA, 3 months|
|9||62/M||Palmoplantar pustulosis||Soles||–||nb-UVB 2 years|
|10||50/F||Palmoplantar pustulosis + Arthropathy||Palms, soles||lt. shoulder joint, sternoclavicular joint, elbow joints, wrist joints||None|
|11||52/F||Palmoplantar pustulosis + Arthropathy||Lt. sole||Shoulder joints, knee joints, wrist joints, ankle joints||CyA (100 mg) 3 years|
|12||38/M||Palmoplantar pustulosis||Palms, soles||–||None|
|13||58/M||Palmoplantar pustulosis + Arthropathy||Palms, soles||Sternoclavicular joint||None|
|14||39/M||Palmoplantar pustulosis + Arthropathy||Palms, soles||Sternoclavicular joint, waist||Biotin, PUVA, mynocycline, loxsoprofen (60 mg) 3 tab/day, 1 month|
|15||58/F||Palmoplantar pustulosis + Arthropathy||Legs, palms, soles||Rt. ankle joint||Biotin|
|16||56/F||Palmoplantar pustulosis + Arthropathy||Palms, soles||Shoulder joints, sternoclavicular joint, waist, wrist joints, lt. finger joint, toe joints||Mynocycline (100 mg), loxsoprofen (60 mg) 1tab, single use|
|17||61/F||Palmoplantar pustulosis + Arthropathy||Palms, soles||Shoulder joints, sternoclavicular joint, wrist joints, finger joints, waist, hip joints, knee joints, ankle joints||PUVA, etretinate 30–10 mg/day, 6 months|
|18||58/M||Palmoplantar pustulosis + Arthropathy||Palms, soles||Shoulder joints, elbow joints, waist, knee joints||Loxsoprofen (60 mg) 1 tab, acetaminophen (100 mg) 1 tab, single use|
|19||67/M||Psoriatic arthropathy||Trunk, arms, legs||wrist joints, knee joints||None|
|20||24/M||Psoriatic arthropathy||Arms||Finger joints, toe joint, lt. heel||Loxoprofen (60 mg) 1 tab, single use|
|21||26/M||Psoriatic arthropathy||Legs||Rt. shoulder joint, sternoclavicular joint, wrist joints, finger joints, ankle joints, toe joints||None|
|22||45/M||Psoriatic arthropathy||Trunk, arms, legs, buttocks||Knee joints, toe joints, rt. finger joints||Meloxicam (10 mg) 1 cap, single use, loxoprofen (60 mg) 3-1tab/day, 4 years|
|23||56/M||Psoriatic arthropathy||Cubital fossa, palms, soles, hands||Neck, knee joints, finger joints, toe joints, rt. heel||Loxoprofen (60 mg) 3tab/day, 4 weeks|
|24||51/M||Psoriasis vulgaris||Trunk, arms, legs, buttocks||–||CyA, Etretinate, PUVA, nb-UVB|
|25||36/F||Behçet's disease||Lip, tongue||–||Mefenamic acid (250 mg) 1cap/day 5 months, loxoprofen (60 mg) 3tab/day 6 months,|
|26||56/F||Behçet's disease||Rt. labia minora, oral cavity||–||Loxoprofen (60 mg) 1 tab, single use|
|27||23/F||Behçet's disease||Lt. labia majora, oral cavity||–||Loxoprofen (60 mg) 3 tab/day 2 weeks|
|28||36/F||Behçet's disease||Rt. labia minora, oral cavity||Lt. knee joint||Colchicine (0.5 mg) 2 tab/day 1 year, loxoprofen (60 mg) 1 tab, single use|
|29||18/F||Behçet's disease||Oral cavity, back of hands, legs||Knee joints, lt. elbow joint||Loxoprofen (60 mg) 3–2 tab/day 9 months|
|30||25/F||Behçet's disease||Rt. labia majora, oral cavity, lip||–||None|
|31||38/F||Behçet's disease||Lip, oral cavity||Lt. knee joint, lt hip joint||Colchicine (0.5 mg) 2 tab/day 2 years|
|32||39/F||Behçet's disease||Lt. labia majora||Lt. knee joint, lt. elbow joint||Prednisolone 30–10 mg/day, 6 months, Colchicine (0.5 mg) 2 tab/day 6 months|
|33||74/M||Behçet's disease||Perianal||–||Loxoprofen (60 mg) 1tab, single use|
|34||37/M||Pyoderma gangrenosum||Bilateral axillae, back, buttocks||–||None|
|35||37/M||Pyoderma gangrenosum||around artificial anus||–||None|
|36||49/M||Pyoderma gangrenosum||Rt. lower leg||–||None|
|37||76/M||Pyoderma gangrenosum||Rt. lower legs||–||None|
|38||21/M||Pyoderma gangrenosum||Neck, face, trunk||–||Dapsone (25 mg) 3T/day, 1 month, PSL (5) 6T-3T/day, 6 months|
|39||27/F||Pyoderma gangrenosum||Lower legs||–||None|
|40||73/M||Reiter's disease||Penis, scrotum, rt. groin, rt. hand, rt. arm, rt. chest||Shoulder joints, elbow joints, waist, knee joints||Diclofenac (25 mg) suppository 1 tab, single use|
|41||21/F||Acrodermatitis continua of Hallopeau||Palms, fingers||–||PUVA, CyA 250 mg/day, 7 days|
|42||34/M||Cutaneous allergic vasculitis||Arms, legs||Ankle joints||Prednisolone 20–10 mg/day, sporadically.|
|43||65/M||Sweet's syndrome||Back, lt. palm, lt. knee||Lt. shoulder, waist, hip joints, lt. knee||Loxoprofen (60) 1tab, single use|
|44||66/M||Erythema nodosum||Rt. leg, knees, rt. arm||–||Cefotaxime|
Granulocyte and monocyte adsorption apheresis is an extracorporeal-type apheresis unit that uses a column containing 3 × 104 pieces of 2 mm-diameter cellulose acetate beads. In the current series, GMA was performed 5 or 10 times at 5-day intervals. If the lesion showed amelioration but persisted at the completion of the 5th treatment, five more treatment sessions were added. In each session, 1800 mL of blood are drained from the cubital vein of one arm, passed at a flow rate of 30 mL/min through the GMA column, and returned to the cubital vein of the other arm. The GMA column, its circuit lines, and circuit system consisting of a regular dialysis circuit and a pump, were provided by Japan Immunoresearch Laboratories (Takasaki, Japan). GMA was performed without adding any other systemic remedy; in patients who already received medication, the dose was left unchanged. Concomitant medications and their duration prior to GMA are shown in Table 1. GMA was carried out after obtaining the prior written informed consent of all participants. All procedures conducted in GMA therapy were approved by the Human Investigation Committee of Kagoshima University Graduate School of Medical and Dental Sciences.
Clinical and laboratory evaluation
The severity of the patients' skin lesions was evaluated clinically based on overall lesional assessment (OLA)  and recorded on a 5-point scale where 0 = none, 1 = slight, 2 = mild, 3 = moderate, and 4 = severe. For OLA, we considered plaque elevation, erythema, and scaling for PV and PsA; pustules, erythema, and scaling for PP, Reiter's disease, and acrodermatitis continua; area and depth of ulcerations, granulation, and pain for pyoderma gangrenosum, Behçet's disease, and cutaneous allergic vasculitis (CAV); erythema, induration, and tenderness for Sweet's syndrome and erythema nodosum (EN). The response to treatment was rated as excellent in patients who improved by four grades (e.g. grade 4 to grade 0), as good when they improved by two or three grades, and as moderate when improvement was by a single grade; no change and exacerbation were also recorded. All clinical evaluations were made by three independent physicians; if their assessments differed, the median value of their judgment was recorded as the treatment outcome.
The degree of arthropathy, assessed by the patients based on joint pain, the number of affected joints, and the range of joint motion, was represented on a 10-cm visual analog scale (VAS). Treatment responses were recorded as excellent, good, and moderate when the VAS score was 20% or less, 50% or less, and 90% or less, respectively, of the baseline score, or as no change or exacerbation.
The white blood cell (WBC) count, neutrophil count, and C-reactive protein (CRP) were measured before starting GMA therapy and one day after the last treatment session.
Measurement of Mac-1 expression
Flow cytometric analysis of the expression of Mac-1 (integrin CD11b/CD18) on peripheral neutrophils from patients was performed using whole blood and mouse anti-human CD11b antibody (Immunotech, Marseille, France), which is not a blocking antibody, according to the manufacturer's instructions (Beckman Coulter, Fullerton, CA, USA). Briefly, after 30-min incubation of 50 μL of sodium citrate-anticoagulated whole blood with 10 μL of mouse anti-human CD11b antibody, 250 μL of lysis buffer (OptiLyse C; Beckman Coulter) were added. The mixture was incubated for 10 min, 250 μL phosphate-buffered saline (PBS) were added and this was followed by another 15-min incubation. Cells were pelleted by centrifugation at 400 × g for 5 min and analyzed for CD11b expression using an EPICS flow cytometer (Beckman Coulter). All incubations were at room temperature in the dark. Granulocytes were identified by gating on a biparametric histogram represented with side and forward scatter. Mac-1 expression was measured before starting GMA therapy and one day after the last treatment session.
For comparison of the WBC count, neutrophil count, CRP, and Mac-1 expression before and after treatment, Tukey–Kramer's method was used. The results are shown as the mean ± standard error (SE). A difference of P < 0.05 was considered significant.
Response of skin lesions to GMA therapy
We evaluated the skin lesions of 44 patients enrolled in this study. Excellent response was observed in 13 patients; five with PP, three with PG, two with PsA, two with BD, and one with CAV. Good and moderate responses were obtained in 17 and nine patients, respectively. The lesions in five patients (two with PP, two with BD, one with acrodermatitis continua of Hallopeau), were unchanged. In 39 of the 44 evaluated patients (88.6%), the skin lesions showed improvement by GMA therapy (Tables 2, 3).
|Patient no.||Age/Sex||Diagnosis||Treatment sessions||VAS Before/After||Response of skin lesions||Response of arthropathy||Mac-1 (MFI) Before/After|
|5||35/M||Palmoplantar pustulosis + Arthropathy||10||8/4||Moderate||Good||120.6/29.0|
|7||38/F||Palmoplantar pustulosis + Arthropathy||5||3/2||Moderate||Moderate||10.9/7.5|
|10||50/F||Palmoplantar pustulosis + Arthropathy||5||3/1||Excellent||Good||36.5/15.9|
|11||52/F||Palmoplantar pustulosis + Arthropathy||5||3/1||Good||Good||55.9/41.9|
|13||58/M||Palmoplantar pustulosis + Arthropathy||5||2/0||Good||Excellent||11.8/10.3|
|14||39/M||Palmoplantar pustulosis + Arthropathy||5||9/6||Unchanged||Moderate||15.3/15.2|
|15||58/F||Palmoplantar pustulosis + Arthropathy||5||4/1||Good||Good||35.3/37.2|
|16||56/F||Palmoplantar pustulosis + Arthropathy||10||3/1||Good||Good||42.8/29.2|
|17||61/F||Palmoplantar pustulosis + Arthropathy||10||6/1||Good||Good||42.4/23.6|
|18||58/M||Palmoplantar pustulosis + Arthropathy||5||10/3||Excellent||Good||21.9/12.4|
|41||21/F||Acrodermatitis continua of Hallopeau||5||n.a||Unchanged||n.a.||40.7/18.6|
|42||34/M||Cutaneous allergic vasculitis||5||5/0||Excellent||Excellent||n.d.|
|43||65/M||Sweet' s syndrome||5||10/9||Good||Unchanged||24.7/3.1|
|Palmoplantar pustulosis (N = 14)||2||7||3||2|
|Behçet's disease (N = 9)||2||3||2||2|
|Pyoderma gangrenosum (N = 6)||3||1||2|
|Psoriatic arthropathy (N = 5)||2||1||2|
|Pustular psoriasis (N = 4)||3||1|
|Sweet's syndrome (N = 1)||1|
|Psoriasis vulgaris (N = 1)||1|
|Reiter's disease (N = 1)||1|
|Erythema nodosum (N = 1)||1|
|Cutaneous allergic vasculitis (N = 1)||1|
|Acrodermatitis continua of Hallopeau (N = 1)||1|
|Total (N = 44)||13||17||9||5|
|Palmoplantar pustulosis (N = 10)||1||7||2|
|Psoriatic arthropathy (N = 5)||2||1||2|
|Behçet's disease (N = 4)||4|
|Pustular psoriasis (N = 1)||1|
|Cutaneous allergic vasculitis (N = 1)||1|
|Reiter's disease (N = 1)||1|
|Sweet's syndrome (N = 1)||1|
|Total (N = 23)||9||9||4||1|
Representative patients are shown in Figure 1. Patient #4 with generalized PP had irregularly-shaped erythematous lesions with numerous pustules on her trunk and extremities. They had appeared 2 months prior to her visit to our department. The lesions showed dramatic improvement after the first treatment and completely disappeared after the fifth treatment (Fig. 1a). PG in patient #39, which manifested as multiple and painful ulcers on her lower legs, had become exacerbated 2 months before GMA therapy and subsided after 10 treatment sessions (Fig. 1b).
Response of arthropathy to GMA therapy
Arthropathy was evaluated in 23 patients. An excellent response was obtained in nine patients; four with BD, two with PP, two with PsA, and one with CAV. Good and moderate responses were observed in nine and four patients, respectively. The arthropathy of one patient with Sweet's syndrome remained unchanged. Overall, arthropathy improved in 22 of the 23 patients (95.6%) (Tables 2, 3).
The mean values of the WBC and neutrophil count, CRP, and Mac-1 expression level before starting GMA therapy were 7664 ± 728/μL, 5110 ± 702/μL, 2.17 ± 1.26 mg/dL, and 27.1 ± 6.66 MFI (mean fluorescence intensity), respectively. They decreased to 6674 ± 632/μL, 4178 ± 494/μL, 1.14 ± 0.51 mg/dL, and 17.9 ± 3.0 MFI. The decrease in WBC- and neutrophil count and MAC-1 expression level was statistically significant (Table 4).
|WBC (/μL)||7664 ± 728||6674 ± 632*|
|Neutrophils (/μL)||5110 ± 702||4178 ± 494*|
|CRP (mg/dL)||2.17 ± 1.26||1.14 ± 0.51|
|Mac-1 (MFI)||27.1 ± 6.66||17.9 ± 3.02*|
We encountered no adverse events in any of our GMA-treated patients.
The results of the present study, which is open-labeled and uncontrolled, further support our proposal that GMA therapy is effective and safe for the treatment of various skin diseases and associated arthropathy attributable to activated granulocytes. More than 88% of our patients benefited from GMA and no adverse effects were encountered. Inflammatory indices such as the WBC and neutrophil count decreased significantly in response to GMA therapy.
We previously showed that the expression of Mac-1, a heterodimer consisting of CD11b (integrin αM subunit) and CD18 (integrin β2 subunit), on the patients' peripheral neutrophils was increased compared to normal subjects and GMA adsorbs Mac-1 (CD11b/CD18)-expressing neutrophils selectively to the CA beads by the binding of complement component (iC3b) on CA beads and CD11b expressed on activated neutrophils . In accordance with this mechanism, Mac-1 expression on the patients' peripheral neutrophils was significantly decreased in the present study. Our results clearly show that Mac-1 could be a useful biomarker for the neutrophilic diseases. As the results of the removal of activated neutrophils, effector cells of inflammation, serum levels of proinflammatory cytokines including interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-α (TNF-α) were decreased and subsequently clinical manifestations are subsided by GMA therapy . While the mean of Mac-1 expression level decreased significantly in response to GMA therapy, the decrease in MFI did not always correlate with the clinical response in each case. Our study to elucidate the factor responsible for sensitivity of each case is underway.
Based on our favorable clinical experience with GMA therapy and on the in vitro observations presented previously , a multi-centered, prospective, randomized trial of GMA for PP was performed. A total of 15 patients with moderate to severe PP were included. The severity was scored according to the practice guidelines for the treatment of pustular psoriasis, which is proposed by the Research Group for Rare and Intractable Dermatological Diseases funded by the Japanese Ministry of Health, Labour and Welfare. Eligible patients had to have >10% of their skin area to be covered by pustules. Oral etretinate, cyclosporine, methotrexate, prednisolone, and topical prednisolone/vitamin D3 could continue if it was started well in advance of entry. Patients were given five GMA treatment sessions at 6-day intervals. Efficacy was assessed by measuring the skin areas covered in pustules at baseline and 2 weeks after the last treatment session. In this study, one patient did not complete the first treatment session. Based on the PP severity scores relative to entry, the overall scores, the area of erythroderma, pustules, and edema were decreased significantly. Dermatology Life Quality Index (DLQI) was also improved significantly, reflecting better daily function, and quality of life. Good response was obtained in 12 patients (85.7%), and 10 patients maintained clinical response at 10 weeks after the last treatment session without any change in medication . With this excellent outcome of the multi-centered study, GMA has been approved for the treatment of PP by the Japanese Ministry of Health, Labour and Welfare in October 2012.
We propose that granulocyte and monocyte adsorption apheresis is a useful treatment modality for refractory skin diseases attributable to activated neutrophils as well as pustular psoriasis. We are currently engaged in the design of a multi-centered, prospective, randomized trial for other skin diseases of this sort.
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