A Treatment and Monitoring Protocol for Tocilizumab in Refractory Macrophage Activation Syndrome with Systemic Juvenile Idiopathic Arthritis

Summary

This protocol describes the use of tocilizumab, combined with glucocorticoids and cyclosporine A, as salvage therapy for refractory macrophage activation syndrome in children with systemic juvenile idiopathic arthritis.

Abstract

This study evaluated the therapeutic effectiveness and safety of tocilizumab (TCZ) in patients with systemic juvenile idiopathic arthritis (sJIA)-associated refractory macrophage activation syndrome (MAS). A total of 100 patients diagnosed between 2021 and 2022 were included. Patients receiving standard therapy (glucocorticoids plus cyclosporine A) were assigned to the control group (n = 30), while those receiving add-on TCZ were assigned to the study group (n = 70). The treatment protocol included intravenous methylprednisolone pulses followed by oral prednisone tapering, cyclosporine A with trough monitoring, and TCZ administered every 2 weeks. Outcomes included laboratory parameters, cytokine profiles (IL-6, IL-18, IFN-γ, sCD25, sCD163), sJADAS27 scores, clinical response, and adverse events over 24 months. The TCZ group demonstrated faster normalization of laboratory indices and greater reduction in inflammatory cytokines (P < 0.05). Remission rates were higher (71.4% vs. 33.3% at Day 14), with lower recurrence and reduced glucocorticoid exposure. Adverse event rates were lower in the TCZ group (25.7% vs. 46.7%, P < 0.05). These findings demonstrate that TCZ-based combination therapy provides an effective and well-tolerated salvage strategy for refractory sJIA-MAS and support further prospective evaluation of this protocol.

Introduction

Systemic juvenile idiopathic arthritis (sJIA) is an autoinflammatory subtype that represents approximately 10–15% of juvenile idiopathic arthritis (JIA) diagnoses and is characterized by quotidian fever, a transient salmon-colored rash, and arthritis¹. Macrophage activation syndrome (MAS), a life-threatening complication of sJIA, results from excessive T-cell and macrophage activation, leading to a cytokine storm and multi-organ failure². Approximately 10% of patients with sJIA develop overt MAS, with mortality rates reaching 20%–40% in severe cases^3,4. Diagnostic challenges persist, as MAS symptoms often overlap with sJIA flares, and the 2016 classification criteria, despite improved sensitivity, may fail to identify atypical or early-stage cases⁵.

Current treatment strategies for sJIA-associated MAS (sJIA-MAS) center on high-dose glucocorticoids (GC) combined with cyclosporine A (CsA) as first-line therapy⁶. The standard regimen consists of intravenous methylprednisolone pulses (15–30 mg/kg/day for 3 days), followed by oral prednisone (1–2 mg/kg/day), administered concurrently with oral CsA (4–6 mg/kg/day) with trough concentration monitoring⁷. However, this conventional approach has significant limitations. Approximately 30% of patients fail to respond adequately to first-line therapy, and prolonged immunosuppression increases the risk of severe infections, nephrotoxicity, and growth impairment⁸. In patients with an insufficient response, second-line options include etoposide, anakinra, and emerging agents such as emapalumab (an anti-interferon-γ monoclonal antibody) and rapamycin (an mTOR inhibitor), although clinical experience in pediatric MAS remains limited^7,9.

Tocilizumab (TCZ), a recombinant humanized monoclonal antibody targeting the interleukin-6 (IL-6) receptor, is a cornerstone in sJIA treatment¹⁰. Its role in sJIA-MAS remains controversial, as some studies report potential precipitation or exacerbation of MAS^11,12. However, emerging evidence suggests therapeutic benefit in selected populations^13,14. The concept of “inflammatory load stratification,” proposed in a Chinese multicenter study (ChiCTR2300070171), supports early TCZ intervention to prevent an interferon-γ- dominated immune imbalance, although the results remain pending⁸. A recent case report demonstrated that sequential targeted blockade, including ruxolitinib following TCZ failure, achieved rapid remission¹⁵. Long-term follow-up studies further indicate that TCZ can sustain control of inflammation, reduce glucocorticoid exposure, and improve growth outcomes¹⁶.

Significant gaps remain in the management of sJIA-MAS. Reliable predictors of TCZ response or MAS recurrence are lacking. Biomarkers such as soluble cluster of differentiation 25 (sCD25) and soluble cluster of differentiation 163 (sCD163) correlate with disease activity; however, their role in dynamic monitoring remains unclear. Furthermore, interleukin-1 inhibitors, recommended as first-line therapy in Western countries, appear to show limited efficacy in Asian populations^17,18. Ruxolitinib demonstrates therapeutic promise but requires further evaluation of pediatric safety. In addition, even after successful control of MAS, patients may develop sequelae including osteoporosis, growth retardation, and cardiovascular complications, necessitating comprehensive rehabilitation and metabolic management¹⁹.

Based on this background, this retrospective analysis of 100 refractory sJIA-MAS cases comprehensively evaluates the therapeutic effects and safety profile of tocilizumab. Unlike previous studies that focused primarily on single clinical endpoints, this study provides a multi-dimensional evaluation incorporating serial laboratory parameters (hematologic, biochemical, coagulation, and inflammatory markers), dynamic cytokine profiling (IL-6, IL-18, IFN-γ, tumor necrosis factor-α, sCD25, and sCD163), disease activity scoring (sJADAS27), and long-term clinical outcomes including glucocorticoid tapering and recurrence rates. The addition of TCZ to standard therapy (GC + CsA) is evaluated for its potential to improve fever control, accelerate normalization of laboratory indices, and facilitate reversal of organ dysfunction. By systematically tracking the temporal dynamics of inflammatory markers and cytokine networks, this study aims to address the existing evidence gap in targeted therapy for pediatric refractory MAS in China and to inform stratified, precision-based intervention strategies to improve long-term survival and prognosis in this high-risk population.

Protocol

This retrospective study, which analyzed anonymized clinical archives, was approved by the Ethics Committee of the Maternity and Child Healthcare Hospital, Qinhuangdao (Approval No.: QFY000286) in accordance with the Declaration of Helsinki. Due to the study's retrospective nature and the use of pre-existing anonymized clinical data, the ethics committee waived the requirement for informed consent. The reagents, chemicals, and software used are listed in the Table of Materials.

1. Patient selection and cohort formation

1. Screen the electronic medical record system to identify all children diagnosed with sJIA between February 2021 and December 2022. Select patients with a documented complication of MAS.
2. Apply inclusion criteria sequentially:
   1. Confirm the sJIA diagnosis according to the International League of Associations for Rheumatology (ILAR) 2004 classification criteria²⁰.
   2. Verify the MAS diagnosis using the 2016 sJIA-MAS classification criteria²¹. Require documented fever plus serum ferritin > 684 ng/mL and any two of the following: platelet count ≤ 181 × 10⁹/L, aspartate aminotransferase (AST) > 48 U/L, triglycerides > 1.76 mmol/L, or fibrinogen ≤ 3.6 g/L.
   3. Identify patients with insufficient response to initial therapy. Add tocilizumab only in patients who demonstrate an inadequate response to standard first-line therapy.
   4. Define insufficient response as persistent fever (temperature >38.5 °C) and a reduction in serum ferritin of <50% from the pre-treatment peak after ≥7 days of standard therapy.
   5. Define standard therapy as intravenous methylprednisolone pulses (cumulative dose ≥30 mg/kg) combined with oral CsA ≥5 mg/kg/day²².
   6. Initiate tocilizumab immediately after confirming insufficient response, typically within 24–72 h after Day 0 assessment.
      NOTE: Anakinra (an interleukin-1 inhibitor) was not used in this cohort due to limited availability in China during the study period (2021–2022) and lack of regulatory approval for pediatric use in this indication. Consequently, CsA and tocilizumab represented accessible second-line options for the management of insufficient response.
   7. Ensure patient age is between 2 and 16 years and disease duration is ≥3 months at the time of insufficient response.
3. Apply exclusion criteria:
   1. Exclude patients with active documented infections, including bacterial (positive blood culture), viral (positive Epstein–Barr virus or cytomegalovirus PCR), fungal infections, active tuberculosis, or human immunodeficiency virus seropositivity.
   2. Exclude patients with malignancies, congenital immunodeficiency disorders, severe heart failure (New York Heart Association class III–IV), or liver cirrhosis (Child–Pugh class B or C).
   3. Exclude patients with severe cytopenia (absolute neutrophil count <1.0 × 10⁹/L or platelet count <50 × 10⁹/L) or severe hepatic injury (alanine aminotransferase (ALT) or AST >10 × ULN).
   4. Exclude patients who received biologic agents (e.g., anti-interleukin-1, anti-interleukin-6, or Janus kinase inhibitors) within 3 months prior to refractory MAS onset.
   5. Exclude patients with >20% missing critical data (e.g., serial ferritin measurements, glucocorticoid dosing records).
4. Form treatment groups:
   1. Categorize patients into two groups based on treatment after fulfilling refractory MAS criteria.
   2. Define control group: patients receiving only glucocorticoids and CsA.
   3. Define study group: patients receiving tocilizumab in addition to glucocorticoids and CsA (Figure 1).

2. Administration of combined immunomodulatory therapy

1. Initiate high-dose glucocorticoid therapy²³:
   1. Administer intravenous methylprednisolone 15–30 mg/kg/day (maximum 1 g/day) for 3 days.
   2. Transition to oral prednisone 1–2 mg/kg/day in 2–3 divided doses (maximum 60 mg/day).
2. Initiate and monitor CsA therapy:
   1. Administer oral CsA at 4–6 mg/kg/day in two divided doses.
   2. Monitor trough concentrations regularly.
   3. Adjust dosage to maintain 100–200 ng/mL.
3. Prepare and administer TCZ.
   1. Initiate TCZ within 24–72 h after confirmation of insufficient response.
   2. Calculate dose: <30 kg → 12 mg/kg; ≥30 kg → 8 mg/kg.
   3. Dilute in 100 mL sterile 0.9% sodium chloride.
   4. Infuse intravenously over ≥60 min.
   5. Repeat every 2 weeks.
4. Establish monitoring schedule:
   1. Perform weekly monitoring of complete blood counts, ALT, AST, and renal function.
   2. Reduce monitoring to every 2 weeks after stabilization.
5. Implement glucocorticoid tapering:
   1. Initiate tapering after MAS control (no fever for ≥72 h and ferritin reduction >50%).
   2. Reduce prednisone by 0.2 mg/kg/day every 7–10 days until 0.5 mg/kg/day.
   3. Further reduce by 10% every 2 weeks.
6. Manage toxicity:
   1. Hepatotoxicity: Reduce TCZ by 50% if ALT/AST 3–5× ULN; suspend if >5× ULN; restart at half dose after recovery.
   2. Hematologic toxicity: Suspend TCZ if neutrophils <1.0 × 10⁹/L or platelets <100 × 10⁹/L; resume at reduced dose after recovery.
   3. Infection: Suspend TCZ during serious infection; restart after resolution.
7. Define treatment duration and failure:
   1. Administer ≥4 infusions unless toxicity occurs.
   2. Continue therapy to 12 weeks if response is achieved; consider maintenance every 4 weeks thereafter.
   3. Define failure: persistent fever, no ferritin decline, or organ deterioration after 4 weeks.
8. Definitions of laboratory normalization and remission thresholds:
   1. Define normalization and remission thresholds based on the institutional reference ranges of the Maternity and Child Healthcare Hospital, Qinhuangdao.
   2. C-reactive protein (CRP): normal range < 0.8 mg/dL; remission threshold defined as ≤ 0.8 mg/dL.
   3. Serum ferritin: normal range 20–200  ng/mL; remission threshold defined as < 500  ng/mL based on consensus criteria for MAS resolution.
   4. Fibrinogen: normal range 2–4 g/L; remission threshold defined as > 2 g/L.
   5. Platelet count: normal range 150–450 × 10⁹/L; normalization defined as ≥ 150 × 10⁹/L.
   6. AST and ALT: normal range < 40 U/L; remission threshold defined as ≤ 2 × upper limit of normal (ULN) for resolution of hepatic involvement.
9. Adherence to tocilizumab dosing protocol:
   1. Administer tocilizumab strictly according to the weight-based protocol described in section 2.3.
   2. Permit dose adjustments or treatment interruptions only in the setting of predefined toxicity criteria (hepatotoxicity, hematologic toxicity, or infection) as outlined in section 2.6.
   3. Document all deviations from the protocol, including dose reductions, delays, or premature discontinuation.
   4. Report protocol deviations in the Results section.

3. Data collection and outcome measurement

1. Define Day 0:
   1. Study group: first TCZ infusion.
   2. Control group: eligibility for salvage therapy.
2. Perform baseline assessment:
   1. Conduct assessments prior to Day 0.
   2. Collect demographics, clinical data, laboratory parameters, cytokines, and scores.
3. Schedule follow-up: Day 3, Day 7, Day 14, Day 28, Month 6, Month 12, and Month 24.
4. Collect blood samples:
   1. Collect fasting venous blood.
   2. Process serum samples for cytokine measurement.
      1. Collect blood into serum separator tubes.
      2. Allow samples to clot at room temperature for 30 min.
      3. Centrifuge at 1,500 × g for 15 min at 4 °C within 2 h of collection.
      4. Aliquot separated serum into cryovials and immediately store at −80 °C until analysis.
      5. Do not permit freeze–thaw cycles prior to cytokine measurement.
   3. Process samples for complete blood count and coagulation parameters.
      1. Collect blood into EDTA-containing tubes for complete blood count.
      2. Collect blood into sodium citrate tubes for coagulation parameters (fibrinogen, D-dimer).
      3. Analyze within 4 h of collection.
   4. Analyze hematologic, inflammatory, liver, coagulation, and metabolic markers.
5. Measure cytokines:
   1. Measure using ELISA kits.
      NOTE: IL-6, IL-18, TNF-α, IFN-γ, sCD25, and sCD163 assays were performed according to manufacturer instructions. All cytokine measurements were performed in duplicate for each sample.
      NOTE: To minimize inter-assay variability, serum samples from all time points for each patient were analyzed in the same assay batch whenever possible. For samples that could not be batched, the inter-assay coefficient of variation was maintained below 10% by running quality control standards on each plate. The mean of duplicate measurements was used for statistical analysis.
   2. Quantify IL-6, IL-18, TNF-α, IFN-γ, sCD25, and sCD163.
6. Assess disease activity:
   1. Calculate sJADAS27 scores²⁴ at each time point.
7. Record outcomes:
   1. Record glucocorticoid dose, remission, fever resolution, and recurrence.
   2. Define remission using all criteria²⁵ (fever absence, ≥90% joint improvement, normalized labs).
8. Document adverse events:
   1. Record all events.
   2. Categorize infections, liver dysfunction, cytopenia, allergic reactions, hypertension, and gastrointestinal events.

4. Sample size calculation

1. Perform power analysis using statistical software.
2. Set parameters: d = 0.65, α = 0.05, power = 0.8.
3. Calculate minimum sample size: 88 (26 control, 62 study).
4. Confirm final cohort: 100 participants.

5. Statistical analysis

1. Perform analyses using statistical software.
2. Generate flowcharts using diagram software.
3. Assess distribution: Shapiro–Wilk; Levene’s test.
4. Analyze continuous variables.
   1. Present normally distributed data as mean ± standard deviation (SD) and compare means between two independent groups using the independent samples t-test. Compare means within the same group across time points using a paired-samples t-test.
   2. Present non-normally distributed data as median with interquartile range (IQR) and compare distributions between two independent groups using the Mann–Whitney U test. Compare distributions within the same group across time points using the Wilcoxon signed-rank test.
5. Analyze categorical variables.
   1. Present categorical data as counts and percentages [n (%)].
   2. Compare proportions between groups using the chi-square (χ²) test. Use Fisher’s exact test when any expected cell count is <5.
6. Summarize longitudinal data per time point.
7. Handle missing data.
   1. Exclude cases with missing data on a variable-by-variable basis when missing data are <5% for all key variables. Do not perform data imputation.
8. Define statistical significance.
   1. Consider results statistically significant when the two-sided P-value is <0.05.

Results

A total of 100 children with sJIA-MAS were included in this study, divided into 30 cases in the control group and 70 cases in the study group according to treatment modality. Baseline comparisons demonstrated no statistically significant differences between the two groups in demographic variables (age, sex, body mass index), clinical features (fever, rash, hepatomegaly, splenomegaly, lymphadenopathy, liver dysfunction, encephalopathy, pulmonary edema, circulatory failure, arthritis), laboratory indices (PLT, WBC, NEU, LY...

Discussion

This retrospective controlled analysis demonstrates that tocilizumab (TCZ) combined with glucocorticoids provides significant therapeutic benefit in refractory sJIA-MAS, accelerating normalization of laboratory parameters, promoting clinical remission, and reducing the risks of glucocorticoid dependence and long-term recurrence. A multidimensional evaluation integrating serial laboratory parameters, dynamic cytokine profiling (IL-6, IL-18, IFN-γ, TNF-α, sCD25, sCD163), disease activity scores, and long-term out...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medications
Tocilizumab	Shanghai Roche Pharmaceutical Co., Shanghai, China	S20130020	Recombinant humanized anti-IL-6 receptor monoclonal antibody; 80 mg/4 mL per vial
Methylprednisolone	Pfizer Manufacturing Belgium NV, Puurs, Belgium	H20130301	40 mg per vial; used for intravenous pulse therapy
Cyclosporine A	Roche Pharma (Schweiz) Ltd	S20130020	Immunosuppressant; 80 mg/4 mL oral solution
Blood Collection Materials
Serum separator tubes	Becton, Dickinson and Company, Franklin Lakes, NJ, USA	367983	5 mL, with clot activator and gel separator
EDTA tubes	Becton, Dickinson and Company, Franklin Lakes, NJ, USA	367855	3 mL, K2 EDTA for complete blood count
Sodium citrate tubes	Becton, Dickinson and Company, Franklin Lakes, NJ, USA	363083	3 mL, 3.2% buffered sodium citrate for coagulation tests
Cryovials	Corning Inc., Corning, NY, USA	430659	2 mL, sterile, for serum storage at −80 °C
Cytokine Assay Platform
ProcartaPlex Human Cytokine Panel 1B 25plex	Invitrogen (Thermo Fisher Scientific), San Diego, CA, USA	EPX250-12166-901	Luminex-based multiplex immunoassay; detects IL-6, IL-18, IFN-γ, TNF-α, and 21 other cytokines; requires 25 µL serum
Luminex Instrument	Luminex Corporation, Austin, TX, USA	Luminex 200	For multiplex cytokine detection; xMAP technology
ELISA Kit for sCD25	Boster Bio	EK0400	Sensitivity: 5 pg/mL
ELISA Kit for sCD163	R&D Systems, Minneapolis, MN, USA	DC1630	Sandwich ELISA; detection range: 1.6–100 ng/mL; sensitivity: 0.613 ng/mL; for human serum
ELISA Equipment
Microplate reader	Shanghai Kehua Laboratory System Co., Ltd., Shanghai, China	ST-360	8-channel absorbance reader; filters at 405, 450, 492, 630 nm; reading speed: 5 s/96-well; for ELISA detection
Automatic plate washer	Shanghai Kehua Laboratory System Co., Ltd., Shanghai, China	ST-36W	96-channel plate washer; residual volume ≤1 μL/well; adjustable dispense volume 0–3000 μL; with bottom wash function
Incubator	Shanghai Xin Nuo Instrument Group Co., Ltd., Shanghai, China	EHP-55K	Forced-air convection incubator; temperature range: Amb+5–70 °C; temperature fluctuation ≤±0.2 °C; for ELISA incubation at 37 °C
Adjustable pipettes	Dlab Scientific (Beijing) Co., Ltd., Beijing, China	HiPette/TopPette series	Single-channel pipettes; volumes: 0.5–10 µL, 10–100 µL, 100–1000 µL; for reagent and sample dispensing
Clinical Laboratory Instrumentation
Automated Hematology Analyzer	Mindray, Shenzhen, China	BC-6800Plus	For complete blood count (CBC) including PLT, WBC, NEU, LYM
Chemistry Analyzer	Siemens Healthineers, Erlangen, Germany	ADVIA 1800	For CRP, ESR, liver enzymes (ALT, AST), triglycerides, fibrinogen, D-dimer, ferritin
Coagulation analyzer
ESR measurement system
Statistical Software	IBM SPSS	Version 25.0	Used for all statistical analyses
Diagramming Software	Lucidchart	Online version	Used for generating study flowchart
G*Power Software	Heinrich-Heine-Universität Düsseldorf	Version 3.1.9.7	Used for sample size calculation
Centrifuge	Thermo Fisher Scientific, Waltham, MA, USA	75007201	Sorvall™ ST 8 benchtop centrifuge; for serum separation at 1,500 × g; 4 °C capable
Ultra-low temperature freezer	Thermo Fisher Scientific, Waltham, MA, USA	Forma™900	−80 °C freezer for long-term serum storage
Software
Statistical software	IBM Corp., Armonk, NY, USA	SPSS Statistics version 25.0	Used for all statistical analyses
Sample size calculation software	Heinrich Heine University Düsseldorf, Düsseldorf, Germany	G*Power version 3.1.9.7	Used for power analysis and sample size calculation
Diagramming software	Lucid Software Inc., South Jordan, UT, USA	Lucidchart (online version)	Used for generating study flowchart