Study of the association between nailfold capillaroscopic changes and serum level of interleukin-17 in rheumatoid: a clue for emerging vaculitis

Objectives The aim of this work was to study nailfold capillaroscopic (NC) abnormalities and serum interleukin-17 (IL-17) level among rheumatoid arthritis (RA) patients and to find whether IL-17 is causally involved in the changes in the capillary vascular bed, such as autoimmune prevasculitic changes. Patients and methods The study was conducted on a group of RA Egyptian patients (n=40) who were diagnosed as having RA based on ACR criteria. Those 40 patients were further divided into two groups. Group 1 included RA patients with clinical signs of skin vasculitis and NC changes (n=6). Group 2 included RA patients with no clinical signs of skin vasculitis and no NC changes (n=34). All patients were subjected to demographic data collection, clinical examination, disease activity score 28 calculation, laboratory measurement (including erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, antinuclear antibody, antineutrophil cytoplasmic antibody, anti-cyclic citrullinated peptide, and IL-17) and NC examination. In addition, group 1 (n=6) was further subjected to electrophysiological evaluation using peripheral nerve conduction studies to determine the effect of vasculitis on the peripheral nerves. Results IL-17 level and NC changes showed a significant association in RA vasculitis patients. Conclusion Elevated levels of serum IL-17 and characteristic NC changes raise their importance in the detection of preclinical rheumatoid vasculitis.


Introduction
Rheumatoid vasculitis (RV) is the most serious systemic disease manifestation of rheumatoid arthritis (RA), wherein it manifests almost exclusively in RA patients with rheumatoid autoantibodies and often occurs in the context of other extra-articular manifestations. RV is defined as a clinicopathological manifestation of RA characterized by tissue damage or ischemia verified pathologically by vasculitis [1,2].
(2) An acute or healing arteritis of dermal subcutaneous vessels similar to that seen in polyarteritis nodosa is seen in patients with subcutaneous nodules, livedo reticularis, and ulcers [6,7]. (3) Other histological patterns that can be seen include folliculocentric microabscess formation resembling dermatitis herpitiform and granulomatous vasculitis composed mainly of lymphocytes and histocytes [8].
The postulated mechanisms of vessel wall destruction with RV include the following: [9] (1) Autoantibody targeting of the vessel [10].
RV may involve virtually any organ of the body, but the most common sites of involvement are the skin and peripheral nerves, as in many series the skin or peripheral nerves are involved in more than 80% of RV patients.
Major organ system involvement of the heart, bowel, or kidney is much less common but can lead to significant morbidity and mortality, including myocardial infarction, bowel ischemia, and renal failure; although central nervous system involvement is rare, many case reports describe its occurrence [14,15].  [16,17]. Specialized T cells, called Thelper 17 cells, are the major sources of IL-17 A and IL-17 F in many types of adaptive immunity; however, recently, other contributors to IL-17 A and IL-17-F production, mainly in the innate arm of the immune system, were identified [18].
Although crucial in protecting the host from invasion by many types of pathogens, dysregulated IL-17 production can result in excessive proinflammatory cytokine expression and chronic inflammation, which lead to tissue damage and autoimmunity [19,20].
IL-17 has a role in chronic vascular inflammation of atherosclerosis and possibly hypertensive vascular changes, whereas in acute inflammation IL-17 is elevated and may be causally involved in the autoimmune vasculitides [21][22][23][24][25].
Nailfold capillaroscopy (NC) was used during the beginning of the 20th century, to show in detail the abnormalities that characterize the involvement of microvasculature during Raynaud's phenomenon in systemic sclerosis [26].
NC represents the best method to analyze microvascular abnormalities in autoimmune rheumatic diseases [27].
In addition, capillaroscopic changes have been observed in systemic sclerosis, systemic lupus erythematosis, antiphospholipid syndrome, and Sjogren's syndrome, but still further epidemiological and clinical studies are needed to better standardize the NC patterns [28].

Aim
The aim of this study was to study NC abnormalities and serum IL-17 level among RA patients and to find whether IL-17 is causally involved in the changes in the capillary vascular bed, such as autoimmune prevasculitic changes.

Patients and methods
The current study was conducted on 40 RA patients who were diagnosed according to the current 2010 ACR-EULAR criteria for diagnosis of RA. All patients gave their formal consent. The protocol was approved the Ethical committee of the Alexandria University.
Exclusion criteria included any disease or medication causing vasospasm, any other rheumatic disease, diabetes mellitus, and having hepatitis B or C viruses.
Thereafter, the 40 patients were further divided into two groups based on the presence or absence of NC changes: RA patients with NC changes (group 1; n=6) and RA patients with no NC changes (group 2; n=34).
Thereafter, the six patients included in group 1 were further subjected to electrophysiological studies (in upper limbs: the median nerve and the ulnar nerve; and in the lower limb: the sural nerve and the posterior tibial nerve).

Results
The data collected from patients and controls were tabulated and statistically analyzed to study NC abnormalities and serum IL-17 pattern among RA patients with suspected vasculitis by positive NC changes and those with negative capillaroscopy.
The NC changes that were found in group 1 (n=6) were tabulated.
RA patients with NC changes (group 1) showed clinical evidence of skin vasculitis, which in our studied patients were as follows: three patients suffered from purpuric rash, two patients suffered from small ulcers on the feet and big toe, and one patient suffered from livedo reticularis. All patients included in group 2 with no NC changes showed no clinical manifestations of skin vasculitis.
Patients of group 1 (n=6) showed the same electrophysiological findings and were as follows: absent sural nerve and normal conduction of other nerves.
DAS 28 showed a significant difference between RA patients with no NC changes (n=34) and RA patients with NC changes (n=6) ( Table 1 and Fig. 1). ANA was compared between RA patients with no NC changes (n=34) and RA patients with NC changes (n=6), and it showed no significant difference (Table 3).
ANCA (both types showed the same results) showed a significant difference between RA patients with no NC changes (n=34) and RA patients with NC changes (n=6) (P=0.021) ( Table 3 and Fig. 4).
When IL-17 serum level was compared between RA patients with NC changes (n=6) and RA patients with no NC findings (n=34), a significant difference was found (P<0.001) (Tables 4 and 5 and Fig. 5).

Discussion
The present study included 40 RA patients, of whom only six patients were diagnosed clinically as having RV. This is in agreement with the results of previously published data by Puechal et al. [4], which estimated the prevalence of RV at less than 1-5% in patients and 15-30% in autopsy. Similarly, Loricera et al. [29] reported a low incidence of RV.
In addition, the current study estimated that the incidence of NC changes is the same as the incidence of vasculitis in the studied patients, which shows that NC changes occur in parallel with clinical vasculitic changes. Similarly, Souza and Kayser [30] concluded that NC changes were associated with vascular disorders.
In contrast, Hachulla et al. [31] concluded the opposite, as the results showed that capillaroscopy when compared between RA patients with and those without vasculitis showed no difference, and this was attributed to the fact that the regular capillaroscopy was not accurate in detecting the vascular changes in the nailbed of RV patients.  Box plot showing disease activity score 28 (DAS 28) range in rheumatoid arthritis patients with and without nailfold capillaroscopic changes. This study showed a significant difference in DAS 28 between RA patients and RV patients, and concluded that patients with higher DAS 28 score were associated with vasculitic changes. This means that vasculitis in RA occurs in the disease with high activity as scored with DAS 28 in the studied patients.
Similarly, Shanmugan et al. [32] calculated DAS 28 in RA patients before and after the appearance of Box plot showing the range of C-reactive protein (CRP) among the two groups, rheumatoid arthritis (RA) patients with nailfold capillaroscopic (NC) changes (group 1), and RA patients with no NC changes (group 2).

Figure 3
Box plot showing rheumatoid factor (RF) among the two groups, rheumatoid arthritis (RA) patients with nailfold capillaroscopic (NC) changes (group 1), and RA patients with no NC changes (group 2).  Bar chart showing the level of antineutrophil cytoplasmic antibody (ANCA) between the two groups, rheumatoid arthritis with and without nailfold capillaroscopic changes.  vasculitic ulcer, and it was concluded that there were significant increases in DAS 28 score, which implied a significant difference. In contrast, Mederos et al. [33] concluded that there were discrepancies in data from DAS 28 and recommended to define better score with better cutoff.
In the current study, anti-CCP had no significant difference between RA and RV patients. This implies that anti-CCP is rather a diagnostic than a follow-up tool and its level does not reflect associated vascular abnormalities. In agreement, Wiik et al. [34] concluded that, although anti-CCP is considered an important marker of progressive erosive disease and extra-articular manifestations such as vasculitis, this was hampered by doubt about a positive versus negative reaction in several assays. Korkmaz et al. [35] also concluded that anti-CCP does not seem to have much linkage with extra-articular manifestations of RA.
In contrast, Turesson et al. [36] concluded that anti-CCP levels tend to be higher in patients with RA with vasculitis than in those without vasculitis.
We showed reports that RF showed a significant difference between RA and RV patients, and it was higher in RV patients (P=0.001). This means that RF is highly linked and associated with vascular activity.
Similarly, Khasnis and Langford [37] and Turesson and Matterson [38] concluded that RF level is higher in vasculitis.
In contrast, Tourin et al. [39] proved that RA-associated vasculitis is associated with low titer-positive RF.
The current study illustrated that there was a significant difference in CRP between RA and RV patients, and CRP was higher in the RV group (P=0.010). This reflects the concluded association between CRP and vasculitic changes. Similarly, Suresh [40] concluded the association of high levels of CRP with RV. Moreover, Kobak et al. [41] showed that there was a significant difference in CRP between RA and RV patients.
This is contradictory to that reported by Senolt et al. [42], who showed that CRP can be normal in RA with or without extra-articular manifestations.
The current study concluded that there was no significant difference in ANA between RA and RV patients. This means that ANA is not associated with vasculitic changes in our studied patients.
In agreement, De Souza et al. [43] proved that ANA can be negative, with no significant difference in RV. In contrast, Voskuyl et al. [44] proved that ANA is positive and had significant difference in RV patients.
The current study concluded that ANCA showed a significant difference between RV patients and RA patients (P=0.021). This means that ANCA is associated with vasculitic changes occurring on top of RA in our studied patients.
Similarly, Voskuyl et al. [44] concluded that there is a significant difference in ANCA between RA and RV patients.
In contrast, Puechal [45] and Suresh [40] excluded the possibility of the presence of a significant difference between RA and RV patients.
The current study concluded that there was a significant difference in IL-17 between RV and RA patients, wherein it was more significant in RV patients (P≤0.001). This means that IL-17 is highly associated with the development of vasculitic changes.
In agreement, Hoshino et al. [46] concluded that there is an association between IL-17 and vasculitis. There were no studies to prove otherwise.
Finally, IL-17 and NC changes were proved in our study to have a role in RV.

Figure 5
Box plot representing interleukin-17 (IL-17) level in rheumatoid arthritis patients with and without vasculitis.
Drawbacks in the current study include the following: (a) the sample size of RV patients was small, and (b) RV patients were diagnosed on the basis of the clinical skin evaluation and laboratory measurements, and no biopsy was done.

Conclusion
Elevated levels of serum IL-17 and characteristic NC changes raise their importance in the detection of preclinical RV.

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Conflicts of interest
There are no conflicts of interest.