Original research article/Artykuł oryginalny

Chronic recurrent multifocal ostemyelitis (CRMO) and activityof immune system

Przewlekłe nawracające wieloogniskowe zapalenie kości i szpiku a aktywnośćukładu immunologicznego

Anna Pituch-Noworolska 1,*, Jerzy Sułko 2, Artur Oberc 2

1Department of Clinical Immunology, Polish-American Institute of Pediatrics, Medical College, Jagiellonian University, Cracow, Poland2Orthopedic and Traumatology Division, Polish-American Institute of Pediatrics, Medical College, Jagiellonian University, Cracow, Poland

p e d i a t r i a p o l s k a 8 8 ( 2 0 1 3 ) 3 1 6 – 3 2 2

a r t i c l e i n f o

Article history:

Received: 28.02.2013

Accepted: 21.03.2013

Available online: 26.03.2013

Keywords:� Chronic recurrent multifocal

osteomyelitis� Humoral and cellular immunity� Autoimmunity� Children

Słowa kluczowe:� nawracająca wieloogniskowe

zapalenie kości� odporność humoralna

i komórkowa� autoimmunizacja� dzieci

a b s t r a c t

CRMO (chronic recurrent multifocal osteomyelitis) is a rare inflammatory disease obser-

ved in children. The clinical forms included non-bacterial osteitis acute or chronic, CRMO

and SAPHO (synovitis, acne, pustulosis, hyperostosis and osteitis). The effective therapy

is based on non-steroid anti-inflammatory drugs, steroids and bisphosphonates. The cli-

nical course characterizes remission and relapses. The purpose of this study was analy-

sis of immunological parameters in 9 children with diagnosis of CRMO in remission. In

one patient the co-existance of Crohn's disease was noted. Study included immunoglo-

bulins and C3c, C4 complement molecules level, number of T, B lymphocytes and NK

cells, response to mitogen stimulation. The results showed IgG, IgA and IgM level within

normal range for age, increased level of C3c molecule of complement in 5 children, C4

only in one child. The number of T lymphocytes was increased in 2 patients, decreased

in 3 patients. CD4:CD8 ratio was within normal range in all patients. The number of

activated T lymphocytes (CD3/HLA-DR) was increased in 5 patients. The response to

mitogens stimulation (PHA, PWM and antiCD3 antibody) was decreased in 3 patients.

There were no relations between the initial clinical symptoms, course of disease and

immunological disturbances.

© 2013 Polish Pediatric Society. Published by Elsevier Urban & Partner Sp. z o.o. All

rights reserved.

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/pepo

Introduction

Chronic recurrent osteomyelitis (CRMO) is an inflammatorybone disease affecting mainly children. The inflammation

* Corresponding author at: Katedra Immunologii Klinicznej i Transpla30-663 Kraków, Poland.

E-mail address: mipituch@cyf-kr.edu (A. Pituch-Noworolska).0031-3939/$ – see front matter © 2013 Polish Pediatric Society. Publishhttp://dx.doi.org/10.1016/j.pepo.2013.03.011

presents as bone pain with worsening at night. The labora-tory test show increased parameters of inflammation but inmany patients despite of clinical symptoms these parame-ters are within normal value. The diagnosis is based onclinical symptoms, image assays results especially in MRI.

ntologii, Polsko-Amerykański Instytut Pediatrii, ul. Wielicka 265,

ed by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Table I – Characteristics of patientsTabela I – Charakterystyka pacjentów

Patient 1. B.P. 2. J.G. 3. K.A. 4. O.M. 5. P.K. 6. T.M. 7. W.K. 8. W.E. 9. W.M.

Firstsymptoms

Edema andpain ofleft clavicle

Edema andpain of leftclavicle

Pain of lumbarspine andleft hip

Pain ofright forearm

Pain of right elbow Pain of leftheel andparasternalpart of clavicles

Pain ofthoracicspine

Pain of jaw(right side)

Pain of thoracicspine

Courseof disease

1 year later painof thoracic spine,compressivebreak of Th12

Year later painand edema ofright clavicleand shield bone

Year laterpain of rightleg

2 years later –

relapse,flattening of 2thoracicspinal bones

Pericarditisexudativa

Imagestudies

Bone lysis,periostealreaction

SCI: changesin L1-L5

periostealstratification ofelbow, changesin orbital,jaw, left tightand rightfibula bones,

Histology of biopsy:focal inflammatoryinfiltrationwith neutrophils,lymphocytes, freshconnective tissue,

SCI: changesin leftheel andclavicles

Histology:inflammatoryinfiltrations,SCI: changesin 2nd left rib,Th4–7, Th11,right tight

SCI: changesin jaw

Histology ofTh6-histiocytosis

Effectivetherapy

Azitromycine Azitromycine Azitromycine Azitromycine Azitromycine Azitromycine Azitromycine Bisphosphonates(3 doses)

Bisphosphonates

Additionaldata

Exanthema(SAPHO?)

Psoriasis,acne (SAPHO?)

Steroidswithout effects

Clindamycinewithout effect, Crohndisease treated withsuppression andsteroids

p e

d i

a t

r i

a p

o l

s k

a 8

8 (

2 0

1 3

) 3

1 6

– 3

2 2

317

p e d i a t r i a p o l s k a 8 8 ( 2 0 1 3 ) 3 1 6 – 3 2 2318

The diagnosis is difficult and often delayed because ofparameters suggesting inflammation and therapy with anti-biotics. The clinical course is often supporting this falsediagnosis as remission and temporary improvement ofsymptoms after antibiotics are noted [1–3]. The proposedclassification contains non-bacterial osteitis (NBO), subdivi-ded into acute (ANBO) and chronic (CNBO) forms, chronicrecurrent multifocal osteomyelitis (CRMO) and synovitis,acne, pustulosis, hyperostosis and osteitis (SAPHO) [4, 5].

The etiology of non-bacterial osteomyelitis is unknownalthough the last classifications of primary immunodeficien-cies included these diseases into autoinflammatory syndro-mes [6]. The local and systemic overproduction of pro-inflammatory cytokines (TNF) was noted in active diseasewhat together with typical inflammatory histology [2] sugges-ted such pathomechanism. The diagnosis of CRMO as autoin-flammatory syndrome was leading to use of anti-TNF monoc-lonal antibodies in biological therapy with improvement [7].

The autoinflammatory disorders are characterized byfunctional defects of affected cells and neutrophils are these

Table II – Basic laboratory dataTabela II – Podstawowe dane laboratoryjne

B.P. J.G. K.A. O.M

Parameter

WBC (103/ml) 8.2 7.1 8.7 8.8

Neutrophils (%) 67.9 46.7 61.7 47.8

(103/ml) 5.58 3.3 5.4 4.2

Lymphocytes (%) 17.9 42.5 32.0 43.2

(103/ml) 1.47 3.0 2.8 3.8

Monocytes (%) 13.6

(103/ml) 1.12

Eosinophils (%) 0.4

(103/ml) 0.03

Basophils (%) 0.2

(103/ml) 0.02

Mixed (%) 10.8 6.3 9.0

RBC (mln/ml) 5.78 5.16 4.49 5.02

HGB (g/dl) 15.8 11.0 11.9 12.6

HCT (%) 49.1 37.3 38.1 38.6

MCV (fL) 84.9 72.3 84.9 76.9

PLT (103/ml) 273 485 297 205

CRP (mg/l) 47.5 27.9 29.7 11.1

ALKP (U/L) 146 204.0 190.0 307.0

Na (mmol/l) 142.0 143.6 N.D. N.D.

K (mmol/l) 4.50 5.30 N.D.

Ca (mmol/l) 2.35 1.16 ionized 2.51

Cl (mmol/l) 102.0 102.0 N.D.

Mg (mmol/l) 0.78 N.D. 0.90

P (mmol/l) 1.27 N.D. 1.58

cells in majority of autoinflammatory diseases. In rare formof CRMO (Majeed syndrome) besides neutrophils the hema-topoietic cells are involved as in DIRA syndrome (deficiencyof IL-1 receptor antagonist) monocytes are affected [6].However, in Majeed syndrome the functional defect ofneutrophils is unknown [6]. The prolonged inflammation,the functional defect of neutrophils are often associatedwith other disorders of immune system e.g. high level ofimmunoglobulins, changes in numbers and proportions ofT lymphocytes subpopulations and activations markers.This study analyzed the immunological parameters inchildren with CRMO and SAPHO.

Patients and methods

The study included 9 patients with diagnosis of CRMO andCrohn's disease (one patient). The group consisted 2 boys,7 girls in age from 6 to 16 years (mean value: 13.0 years). Intime of study all patients were in remission without

Patient

. P.K. T.M. W.K. W.E. W.M.

7.4 8.2 9.9 10.2 6.9

61.4 46.5 60.2 57.9 47.1

4.57 3.8 5.9 5.9 3.24

21.8 39.4 29.1 33.2 32.4

1.62 3.2 2.9 3.4 2.23

15.2 15.2

1.13 1.05

1.3 4.9

0.1 0.34

0.3 0.4

0.02 0.03

14.1 10.7 8.9

4.36 5.0 4.91 4.83 4.04

12.1 12.9 13.9 12.7 9.70

38.2 39.7 42.6 39.8 33.1

87.6 79.6 86.8 82.4 81.9

249 289 407 279 517

5.0 N.D. 5.0 12.5 22.1

117.0 N.D. 245.0 205.0 107.0

N.D. N.D. N.D. 142.5 138.0

5.12 4.0

2.51 1.27 ionized 2.22

106.0

N.D. 0.82

Table III – Results of immunological tests in CRMO patientsTabela III – Wyniki testów immunologicznych

Patient

B.P. J.G. K.A. O.M. P.K. T.M. W.K. W.E. W.M.

Parameter data normalvalue

data data data data data data normalvalue

data normalvalue

data normalvalue

Immunoglobulins

IgG (g/l) 15.9 6.38–17.0 14.3 13.2 9.32 14.6 13.1 13.9 15.1 5.83–15.94 24.9 ND

IgA (g/l) 2.6 0.67–3.67 2.9 3.32 1.95 2.01 3.04 1.86 2.96 0.44–2.43 3.6

IgM (g/l) 0.72 0.41–2.03 1.18 1.16 0.91 0.51 0.58 2.30 1.92 0.4–1.93 0.6

Complement

C3c (g/l) 1.16 0.75–1.36 1.41 1.49 1.21 1.37 1.27 1.23 1.46 1.67

C4 (g/l) 0.235 0.13–0.40 0.26 0.326 0.281 0.222 0.206 0.259 0.467 0.289

Lymphocytes

CD3 (/ml) 993 1000–2200 2615 2202 837 1169 1559 2321 2550 1400–3700 792 1881

CD4 (/ml) 602 530–1300 1718 1339 418 827 939 1518 1558 700–2200 480 1302

CD8 (/ml) 316 330–920 710 655 362 288 491 625 850 490–1300 264 517

CD4:CD8 1.9 1.0–3.0 2.4 2.0 1.1 2.8 1.8 2.4 1.8 1.6 2.5

CD3/HLA-DR (/ml) 226 73–162 261 268 113 144 107 744 354 115–215 ND 83

CD19 (/ml) 316 110–570 784 357 113 414 342 357 744 390–1400 96 83

NK (/ml) 180 70–480 261 417 158 198 192 268 213 130–720 300 103

Stimulation index

PHA 52 >20 48 20 15 6 39 13 26 N.D. N.D.

CD3 48 >20 47 25 11 8 30 14 28

PWM 49 >20 41 41 17 17 56 18 27

Neutrophil function present present present present present present N.D. present present

Other data

IgE (IU/ml) 245.0 0.0–200

ANA (titer) (+) 1:500

ASCA (IgA) <20.0 RU/ml (�) 13.3 43.4 (+)

ASCA (IgG) <20.0 RU/ml (+) 41.9 11.2 (�)

p e

d i

a t

r i

a p

o l

s k

a 8

8 (

2 0

1 3

) 3

1 6

– 3

2 2

319

p e d i a t r i a p o l s k a 8 8 ( 2 0 1 3 ) 3 1 6 – 3 2 2320

symptoms of active disease. Clinical symptoms, course ofdisease and response to therapy was shown in Table I.

Methods

Immunoglobulins (IgG, IgA, IgM) level was measured inserum with nephelometry (BNII, Siemens) accordingstandard curve for pediatric patients. The lymphocytessubpopulations, activated T lymphocytes (CD3/HLA-DR),B lymphocytes and NK cells number was assayed withmulticolor flow cytometry (FACSCalibur, Becton-Dickinson)after staining of peripheral blood with monoclonal antibo-dies (Multitest – Becton-Dickinson) in one stage procedurefollowed with erythrocytes lysis. The 5000 or 10,000 eventswere acquired into flow cytometer. The analysis popula-tion of lymphoid cells was performed based on dot plot(CD45 expression versus FSC parameter).

The 3-days culture of isolated lymphoid cells withdensity gradient was performed in the presence of mediumonly (RMPI1640 + 10% FCS), PHA (phytohaemagglutinin), CD3(monoclonal antibodies) and PWM (pokeweed mitogen) asstimulators. After 3 days the H3 Thymidine was added for 6 hfollowed with H3 uptake measurement in beta counter andshowed as cpm (counts per minute). The stimulation of cellswith PHA, CD3 and PWM was calculated as ratio of cpm afterstimulation to cpm in medium. The index value of 20 wasa lower limit of response to stimulation in healthy people.

Results

The studied group of 9 patients with CMRO and Crohn'sdisease (one patient) was observed no less than 2 years. Thedifferent therapy was used but the successful was therapywith azitromycine in 7 patients, bisphosphonates prepara-tions in 2 patients However, the relapses were noted in5 patients including pericarditis exudativa as complicationin one patient (W.M.). This patient was diagnosed ashistiocytosis and treated but the response to this therapywas poor, the relapse occurred and the clinical improve-ment was noted after bisphosphonates. This patient is nowtreated with steroids, azathioprine because of Crohn'sdisease (the diagnosis was based on typical clinical symp-toms, histology of jejunum and presence of antibodies –

antinuclear and ASCA in IgG class).

Basic laboratory tests

The analysis of peripheral blood did not show typical orrepeating changes, The parameters were within normal valuewith one exception (patient W.M.) showing mild anemia (lowvalue of Hgb) and increased number of platelets (Tab. II). Theongoing inflammation process was indicated by increasedvalue of CRP in 6 patients out of 8 patients.

Immunological tests

The IgG level was increased in one patient (W.M.), in remai-ning patients IgG level was within normal with tendency to

higher value, close to upper limit for age (mean value:14.92 g/l). IgA level was increase above upper limit in 2 patients(W.M., W.E.) and close to upper limit in other 2 patients (K.A.,T.M.) (mean value: 2.69 g/l). The level of IgM was increasedonly in one patient (W.K.) (mean value: 1.03 g/l). There wereno repeating pattern of changes in immunoglobulins level.The level of complement C3c molecule was increased in 5 outof 9 patients, the level of C4 was increased in one patient,within normal range in all remaining patients (Tab. III).

The neutrophils function (production of singlet oxygen)was tested with chemiluminescence test after stimulation bylatex. In all patients the response of neutrophils was present.

The cellular immunity was assayed as number ofT lymphocytes, presence and proportions of T lymphocytessubpopulations (CD4+, CD8+), percentage and number ofactivated T lymphocytes (CD3/HLA-DR+), number ofB lymphocytes and NK cells. The function of cells wastested as proliferation index in response to stimulation withmitogens (PHA, PWM) and antigen (CD3).

The number of T lymphocytes (CD3+) was within normalrange in 6 patients, lowered in remaining 3 patients, thesubpopulations CD4:CD8 ratio was preserved in all studiedpatients (normal value according Gent) [8]. The number ofactivated T lymphocytes (CD3/HLA-DR+) was elevated in5 patients (mean value: 370.6/ml), within normal range inremaining 3 patients (mean value: 121.3/ml). ActivatedT lymphocytes consisted from 6.86% (patient T.M.) to 32.05%(patient W.K.) of T lymphocytes. The increased number ofCD3/HLA-DR+ T lymphocytes might suggest the permanentactivation and stimulation of T lymphocytes by ongoingautoimmune process.

The B lymphocytes number was elevated in 1 patient,decreased in 1 patient and normal in remaining patients. TheNK cells number was within normal range in all patients.

The results of lymphocytes response to stimulationshowed normal value of stimulation index in 5 patients outof 8 tested, lower value of index in remaining 3 patients.There were no signs of activation of lymphocytes showed ashigh value of spontaneous proliferation in the culturein vitro. There were no association between number of totalT lymphocytes, activated T lymphocytes and response tostimulation. The lymphocyte response to stimulation andnumber of activated T lymphocytes were not tested in W.M.patient because of immunosuppression (azathioprine) astherapy for active Crohn disease.

Discussion

The results of immunological tests in 9 patients withdiagnosis of CRMO in remission showed mainly disturbancesin cellular immunity. The low number of T lymphocytes(3 patients), increased number of T lymphocytes (2 patients),increased number of activated T lymphocytes (CD3/HLA-DR)(5 patients) and low response to stimulation (3 patients)were noted despite of lack of active disease. The ratio ofT lymphocytes subpopulations was preserved within normalrange. Another interesting observation was increased levelof C3c molecule of complement in 5 patients and C4 in onepatient. There was no unique pattern of changes in studied

p e d i a t r i a p o l s k a 8 8 ( 2 0 1 3 ) 3 1 6 – 3 2 2 321

parameters of immune system. Observed disturbancesindicated ongoing process of inflammation (activatedT lymphocytes, complement molecules) despite of lack ofclinical symptoms of active disease. The increased level ofIgG, complement, low level of T and B lymphocytes andpresence of autoantibodies in patient with diagnosis ofCrohn's disease are associated with process of autoimmu-nity/autoinflammation in active disease. Therapy of thispatient included steroids, azathioprine and sulfosalazine.

The including of CRMO into autoinflammatory diseasesgroup [6] means the chronic, prolong inflammation process.Within our patients observed in remission without clinicalsymptoms of active disease the immunological parameterssuggesting onging inflammatory process were noted in 5 ofthem as increased number of activated T lymphocytes andincreased level of C3c molecule of complement. However,the analysis of total number of T lymphocytes showednormal value, slight increase or decrease of T lymphocytesnumber. The decrease of T lymphocytes, activatedT lymphocytes and B lymphocytes number in patients withdiagnosis of Crohn's disease is explained by suppressivetherapy. The co-existance of CRMO and Crohn's disease issuggesting the common background and pathomechanisme.g. autoinflammation [5, 9–13]. The analysis of CARD15 genevariant associated with Crohn disease did not support thehypothesis of common gene background of CRMO andCrohn's disease. From the other side this study suggestedthat Crohn's disease observed in patients with CRMOrepresenting the extended phenotype of Crohn's disease[13]. In these group CRMO was preceding onset of Crohn'sdisease in 3 patients, the parallel occurrence of bothdiseases was observed in one boy. Histology of jejunumbiopsy showed typical features, no difference betweenpatients with CRMO and without this disease [13].

The co-existance of CRMO and Crohn's disease mightsuggest the different response of inflammmatory jejunumdiseases to typical therapy. In case of 29 years old man theCrohn's disease was resistant to standard therapy (mesala-zine, azathioprine) with improvement after infliximab the-rapy. This therapy with monoclonal antibody was effectivenot only for symptoms of Crohn's disease but also for femurpain as main symptom of active CRMO [11]. Influence oftherapy used because of jejunum inflammatory disease inpatient with CRMO on symptoms of CRMO was showed in12 years old girl with ulcerative colitis and CRMO. After verysevere clinical course of colitis the surgical therapy led torecovery from colitis and prolonged remission of CRMO [14].These cases are supporting the hypothesis of sharing com-mon pathomechanism between CRMO and inflammationjejunum diseases but till now it was not demonstrated andproved. Moreover, the explanation of co-existance of CRMOand jejunum inflammatory disease based on common patho-mechanism might help in finding the effective therapy forboth diseases.

Conclusions

In patients with CRMO the immunological disturbances arenoted mainly within T lymphocytes population. There is no

correlation between the clinical symptoms, course of disease,therapy response and disturbances of cellular immunity.

Authors' contributions/Wkład autorów

AP-N – study design, data interpretation, acceptance of finalmanuscript version, literature search. JS – study design, datacollection, statistical analysis, acceptance of final manusc-ript version. AO – data collection, statistical analysis.

Financial support/Finansowanie

None declared.

Conflict of interest/Konflikt interesu

None declared.

Ethics/Etyka

The work described in this article have been carried out inaccordance with The Code of Ethics of the World MedicalAssociation (Declaration of Helsinki) for experiments invol-ving humans; EU Directive 2010/63/EU for animal experi-ments; Uniform Requirements for manuscripts submitted toBiomedical journals.

r e f e r e n c e s / p i �s m i e n n i c t w o

[1] Ferguson PJ, Sandu M. Current understanding of thepathogenesis and management of chronic recurrentmultifocal osteomyelitis. Curr Rheumatol Rep 2012;14:130–141.

[2] Gikas PD, Islam L, Aston W, Tirabosco R, Saifuddin A, BriggsTWR, et al. Nonbacterial osteitis: a clinicalhistopathological and imaging study with a proposal forprotocol-based management of patients with thisdiagnosis. J Orthop Sci 2009;14:505–516.

[3] Wipff J, Adamsbaum C, Kahan A, Job-Deslandre C. Chronicrecurrent multifocal osteomyelitis. Joint Bone Spine2011;78:555–560.

[4] Jansson A, Renner ED, Ramser J, Mayer A, Haban M, MeindlA, et al. Classification of non-bacterial osteitis.Rheumatology 2007;46:154–160.

[5] Tlougan BE, Podjasek JO, O'Haver J, Cordova KB, Hguyen XH,Tee R, et al. Chronic recurrent multifocal osteomyelitis(CRMO) and synovitis,. Acne, pustulosis, hyperostosisand osteitis (SAPHO) syndrome with associated neutrophilisdermatoses. Pediatr Dermatol 2009;26:497–505.

[6] Al-Herz, Bousfiha A, Casanova J-L, Chapel H, Conley ME,Cunnigham-Rundles C, et al. Primary immunodeficiencydiseases. Front Immunol 2011;2:54.

[7] Eleftheriou D, Gerschman T, Sebire N, Woo P, Pilkinton C,Brogan PA. Biologic therapy in refractory chronicnon-bacterial osteomyelitis of childhood. Rheumatology2010;49:1505–1512.

[8] van Gent R, van Tilburg CM, Nibbelke EE, Otto SA, Gaiser JF,Janssens-Korpela PL, et al. Refined characterization and

p e d i a t r i a p o l s k a 8 8 ( 2 0 1 3 ) 3 1 6 – 3 2 2322

reference values of the pediatric T- and B-cellcompartments. Clin Immunol 2009;133:95–107.

[9] Carpenter E, Jackson MA, Friesen CA, Scarbrough M, RobertsCC. Crohn's-associated chronic recurrent multifocalosteomyelitis responsive to infliximab. J Pediatr2004;144:541–544.

[10] Bousvaros A, Marcon M, Treem W, Waters P, Issenman R,Couper R, et al. Chronic recurrent multifocal osteomyelitisassociated with chronic inflammatory bowel disease inchildren. Dig Dis Sci 1999;44:2500–2507.

[11] Bret J, Lemaire O, Prat C, Zabraniecki L, Fournie B. Asepticosteomyelitis responding to TNF a antagonist therapy in a

patient with Crohn's disease. Joint Bone Spine 2008;75:489–491.

[12] Bognar M, Blake W, Agudelo C. Chronic recurrent multifocalosteomyelitis associated with Crohn's disease. Am J MedSci 1998;315:133–135.

[13] Morbach H, Dick A, Beck C, Stenzel M, Muller-HermalinkHK, Raab P, et al. Association of chronic non-bacterialosteomyelitis with Crohn's disease but not with CARD15gene variants. Rheumatol Int 2010;30:617–621.

[14] Bazrafshan A, Zanjani KS. Chronic recurrent multifocalosteomyelitis associated with ulcerative colitis: a casereport. J Pediatr Surg 2000;35:1520–1522.