ORIGINAL ARTICLE

Comparison of serological markers between ACPA+

and ACPA2 of RA patients

Chunhua Xun • Yong Zhao

Received: 18 August 2010 / Accepted: 30 December 2010 / Published online: 19 January 2011

� Springer-Verlag 2011

Abstract Anti-citrullinated protein/peptide antibodies

(ACPA) have recently emerged as sensitive and specific

serological markers of rheumatoid arthritis (RA), providing

superior alternative of the rheumatoid factor (RF) test in

the laboratory diagnostics of RA. We compare the change

of serum RF, CRP, IgG, IgM, IgA, total complement, C3

and C4. The sera sample was collected from 123 patients

with RA. ACPA were detected with ELISA, and RF, CRP

and total complement (Ct), C3 and C4 were examined by

automatic biochemical analyzer. Serum RF and total

complement concentrations were significantly higher in

ACPA? than in ACPA-, but there were no correlation

between ACPA and RF and Ct. Between ACPA? and

ACPA-, there were no significant difference of CRP, IgG,

IgM, IgA, total complement, C3 and C4. While there were

significant correlation between the concentration of C3 and

IgM and ACPA in ACPA?. Conclusion: This is the first

study to show that ACPA concentration in ACPA?

patients with RA is positively related to serum IgM and C3

levels.

Keywords ACPA � RA � RF � Total complement �C3 � IgM

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory sys-

temic disorder with an autoimmune pathogenesis. With

significantly improved therapy options now available, early

treatment could be shown to prevent irreversible joint

damage reducing signs and symptoms of erosion and

improving physical function. This requires a reliable

diagnosis in the early stages of disease precipitation, since

a substantial number of patients can undergo spontaneous

remission. Rheumatoid arthritis–associated autoantibodies

are helpful serological tools in stating the definite diagno-

sis. A plethora of relevant autoantibodies has been

described in RA patients [1–4]. The presence of rheuma-

toid factor (RF) is included as the laboratory criterion of

RA in the 1987 revised American College of Rheumatol-

ogy (ACR) criteria [5]. However, RF is not specific for this

disease and can also be detected in other rheumatic disor-

ders, especially connective tissue diseases, other chronic

inflammatory diseases, infections, and even in healthy

individuals, particularly the elderly [6]. Anti-citrullinated

peptides antibodies were first described by Schellekens

et al. [7]. These components were determined by ELISA

based on highly purified synthetic linear peptides contain-

ing modified arginine residues (citrulline) serving as anti-

gen [8–10]. These cyclic variants of citrullinated peptides

were used as antigen in the first-generation CCP test [11];

the currently available second-generation tests use highly

reactive peptides, identified from dedicated libraries of

citrullinated peptides, screened with RA sera [12]. Recent

studies indicate that the CCP2 test is very specific and

sensitive for RA [13–15].

More and more studies showed that Anti-citrullinated

protein/peptide antibodies (ACPA) could play an important

role in RA. In this study, we compare serological markers,

C. Xun (&)

Laboratory Department, The affiliated hospital of Jiu Jiang

University, Jiujiang 332000, Jiangxi, China

e-mail: z6x2m@126.com

Y. Zhao

Basic Medical Sciences of Jiu Jiang University,

Jiujiang 332000, Jiangxi, China

123

Rheumatol Int (2012) 32:1143–1146

DOI 10.1007/s00296-010-1757-y

such as RF, CRP, IgG, IgM, IgA, total complement, C3 and

C4, between ACPA? and ACPA- of RA patients and seek

the association with ACPA level and serological markers,

which would try to find a potential way to elucidate the

functional mechanism of ACPA.

Materials and methods

Study population

A total of 123 patients were diagnosed as RA according to

the ACR criteria [5] and constituted the RA study popu-

lation [35 men (28%) and 88 women (72%); median age:

56 years (range: 20–79 years)]. Blood was taken to a

serum tube from an antecubital vein. All serum specimens

were aliquoted within 5 h following sampling; they were

stored at -80�C and thawed only once before being

assayed with all the kits investigated. The study was

approved by our hospital ethics committee. Patients’

informed consent for the usage of sera was obtained prior

to sample collection.

Methods for serologic markers’ determination

ACPA were detected with commercially second-generation

ELISA (Euro-Diagnostica, The Netherlands) according to

the manufacturers’ instructions. The concentrations of RF,

CRP, IgG, IgM, IgA, total complement (Ct), C3 and C4 were

quantified using automatic biochemical analyzer (AU2700,

Japan). ACPA \ 25 U/mL and RF \ 10.3 IU/mL were

considered as negative.

Statistical analysis

Statistical analysis of the results was carried out by using

McNemarv2 test, t-test, and Pearson correlation analysis

with SPSS13.0 software. Values were expressed as

mean ± S. P values \ 0.05 were considered significant.

Result

Examination of ACPA and RF

There were 89 ACPA? and 34 ACPA- among 123 RA

patients’ sera. ACPA- and RF-positive percentages were

72.4% (89/123) and 70.7% (87/123), respectively. There

were only 4 cases of RF \ 10.3 IU/mL in 89 ACPA- and 2

cases of RF [ 10.3 IU/mL in 34 ACPA-. ACPA-positive

percentage was higher than RF, but no significant difference

was found by McNemarv2 test (Table 1).

Comparison of serologic markers between two groups

No significant difference was found in the concentrations

of CRP, C3, C4, IgG, IgM, IgA (P [ 0.05) between

ACPA? and ACPA-. But RF and total complement levels

were significantly higher in ACPA? than in ACPA-

(P \ 0.05) (Table 2).

Correlation analysis

In ACPA? group, there were no significant correlation

between ACPA level and the concentrations of RF, CRP, Ct,

C4, IgG, IgA (P [ 0.05), but ACPA level has significant

association with C3 and IgM levels (P \ 0.05) (Table 3).

Table 1 ACPA and RF statistical analysis

RF ACPA Sum McNemarv2 test

? - v2 P

? 85 2 87 0.167 0.687

- 4 32 36

Sum 89 34

Table 2 Comparison of serologic markers between ACPA? and

ACPA- by independent t test

Group ACPA? ACPA- P value

RF (U/mL) 481 ± 681 17.68 ± 24.4 0.000

CRP (mg/L) 33.09 ± 73.65 33.78 ± 46.09 0.788

Ct (U/mL) 65.25 ± 6.73 61.33 ± 6.18 0.006

C3 (g/L) 1.33 ± 0.26 1.24 ± 0.25 0.096

C4 (g/L) 0.29 ± 0.11 0.27 ± 0.12 0.616

IgG (g/L) 16.73 ± 5.01 16.27 ± 5.05 0.630

IgM (g/L) 3.57 ± 1.66 3.67 ± 1.81 0.927

IgA (g/L) 1.67 ± 0.86 1.57 ± 0.92 0.637

Table 3 Correlation analysis between ACPA and other biological

markers

Group mean ± SD CO P value

CCPA (U/mL) 452 ± 398

RF (U/mL) 418 ± 681 0.162 0.101**

CRP (mg/L) 33.09 ± 73.65 0.082 0.393**

Ct (U/mL) 65.25 ± 6.73 0.032 0.782**

C3 (g/L) 1.33 ± 0.26 0.292 0.002**

C4 (g/L) 0.29 ± 0.11 0.100 0.300**

IgG (g/L) 16.73 ± 5.01 0.088 0.374*

IgM (g/L) 3.57 ± 1.66 0.198 0.046*

IgA (g/L) 1.67 ± 0.86 0.074 0.458*

CO Correlation coefficient

Comparison to ACPA, * P \ 0.05 was considered significant

** P \ 0.01 was considered significant

1144 Rheumatol Int (2012) 32:1143–1146

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Discussion

The presence of several citrullinated proteins has been

demonstrated in the RA synovium. The determination of

ACPA may have important prognostic significance. ACPA

production has been associated with several genetic pre-

disposing factors, including HLA-DRB1 and PTPN22

1858T alleles, as well as with environmental and lifestyle-

related factors, primarily smoking and possibly, the use of

oral contraceptives, and excessive caffeine intake [16, 17].

One recent study has shown that ACPA can trigger Fc

receptors [18]. Some findings indicate that IgM antibody

can profoundly influence immune responses and suggest

that some of these effects are mediated by binding to

effector molecules such as Fc receptor (FcR) and com-

plement via its carboxyl constant regions [19]. The com-

plement system can be activated via 3 pathways: the

classical pathway, the lectin pathway, and the alternative

pathway [20]. Each pathway is initiated by a specific rec-

ognition molecule. The classical pathway is initiated by

C1q; the alternative pathway is initiated by C3. Initiation of

complement activation via each of these pathways results

in the formation of C3 convertases that cleave C3 to pro-

duce biologically active complement fragments that result

in opsonization, chemotaxis, and cytolysis. Complement is

present in synovial fluid and is partly produced locally and

partly originates from the circulation [21]. Complement

levels in the synovial fluid of RA patients can be lower than

those in controls because of local consumption [22]. But

complement activation products have been shown to be

increased in synovial fluid [23]. Similarly, Our data showed

that serum total complement level was higher in ACPA?

than in ACPA-, while no association was showed between

ACPA and total complement level, but there was a positive

correlation between ACPA and C3 level. Now that ACPA

are increasingly recognized as major players in RA; it is of

relevance to know if and how these antibodies activate

complement. Although one could expect that ACPA can

activate the complement system, not all antibody isotypes

do so to the same extent, and antibodies from different

patients may use different pathways for activation. IgM,

IgG3, and IgG1 are the most potent complement-activating

isotypes. The ACPA response involves all IgG subclasses

as well as IgM and IgA [24, 25]. ACPA found in patients

with arthritis can activate the human complement system

via both the classical and the alternative pathways [26].

In conclusion, ACPA can activate effector mechanisms of

the immune system largely via 2 pathways, namely, by

binding to IgM Fc receptors and by activation of the

complement system.

Up to now, no associated report was found about the

correlation between serum ACPA and biological markers

in ACPA? patients with RA. We detected serum ACPA

with ELISA method in 123 RA patients; meanwhile, the

concentrations of RF, CRP, IgG, IgM, IgA, total comple-

ment (Ct), C3 and C4 were examined using automatic

biochemical analyzer. The diagnostic sensitivities of RA

with ACPA and RF were 70.7 and 72.4%, respectively,

thus almost matched former reports [3]. RF and total

complement showed significantly higher correlation in

ACPA? than in ACPA-, whereas in ACPA? group, there

were no significant correlation between ACPA and RF,

total complement, but there were significant correlation

between ACPA and C3, IgM. Our data indicate that ACPA

are capable of activating a major immune effector includ-

ing C3 and IgM via a certain pathway. Larger studies

would verify the results, and further studies would proceed

to elucidate the surprising phenomena.

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