Humoral and cellular immune response to Buerger’s disease

Abstract

Objective

Thromboangiitis obliterans is a nonatherosclerotic occlusive disease, affecting small to moderate sized arteries of the upper and lower extremities, leading to progressive inflammation and clot formation. However, the role of humoral and cell-mediated immunity in the development of this disease has not been clearly identified. The present study was intended to investigate the humoral and cellular immune response in patients with Buerger’s disease with different disease severity.

Methods

In an observational study, 80 male patients with Buerger’s disease were included and categorized into three groups (mild, moderate, and severe) based on clinical manifestations. After blood sampling, cellular phenotypes were determined, and erythrocyte sedimentation rate, immunoglobulins (Ig) A, M, G, and E, as well as C3 and C4 components of the complement system and complement hemolytic activity (CH50) were measured.

Results

The mean age of the patient was 42.85 ± 8.39 years. Pulse abnormality, cold intolerance, and claudication were the most common symptoms. Eleven (13.75%), 46 (57.50%), and 23 (28.75%) patients had mild, moderate, and severe symptoms. Regression analyses showed that the presence of severe symptoms was significantly associated with elevated erythrocyte sedimentation rate and C4 levels (p < 0.05).

Conclusion

Buerger’s disease in severe cases was associated with increased erythrocyte sedimentation rate and abnormal C4 levels. The alterations in these inflammatory biomarkers might be due to a secondary inflammatory response to the presence of ulcer or gangrene and the inflammatory process in patients with severe symptoms.

Keywords

Thromboangiitis obliterans severity immune system immunoglobulins complement system proteins

Introduction

Thromboangiitis obliterans (TAO) or Buerger’s disease is a nonatherosclerotic occlusive disease of small to moderate vessels (arteries and veins) of upper and lower extremities.^1,2 The occlusion is caused by the progressive inflammation and clot formation.^1,3 This disease is commonly associated with tobacco exposure, especially cigarette smoking.³ However, it has been also reported with the use of smokeless tobacco.⁴ Abstinence from tobacco is the only definitive treatment of Buerger’s disease.^3,5,6

Buerger’s disease usually presents with ischemic pain and ulcerations of the fingers and toes in young male smokers with the involvement of distal extremity vessels.^3,4 However, large vessel involvement has been reported less frequently and may lead to amputation.^7,8 Two or more limbs are usually involved in patients with Buerger’s disease, with thrombophlebitis and Raynaud’s phenomenon being observed in roughly 40% of them.^8,9

The prevalence of Buerger’s disease is increasing in the Central Asia and Far East, despite the decreasing occurrence in North America and Western Europe.^3,5 Smoking has been identified to play a pivotal role in the incidence and pathogenesis of Buerger’s disease.^4,5 However, there are articles in the literature reporting patients who were not smokers.^10,11 Various studies have been conducted to evaluate the relationship between the humoral and cellular immune response and development or progression of Buerger’s disease.^1,10,12

The immune system is responsible for distinguishing self from non-self molecules.¹³ The acquired immunity via B lymphocytes and T lymphocytes is, respectively, known as humoral and cell-mediated immunity. However, this categorization is not absolute, and humoral and cellular immune responses are interrelated.^3,13

The etiology of Buerger’s disease has been considered to be associated with autoimmunity and the effects of tobacco on the immune system.^4,8 Some studies have reported interferon reduction, decreased activity in the provision of antigens and neutrophils, decreased circulating immunoglobulins, and inhibition of cytokines; all of which reduce the acquired and inherent immunity and lead to susceptibility to infections. These findings indicate the role of the immune system in the development of this disease.^1,14 However, there are still contradictions in the results of these studies. Therefore, the present study was intended to evaluate the role of the humoral and cellular immune responses to Buerger’s disease.

Materials and methods

Patient selection

We had established a regional data registry, including 242 patients with or suspicious for Buerger’s disease. After careful evaluation of patients, the diagnosis of Buerger’s disease was confirmed in 225 cases. Of these 225 patients with Burger’s disease, 91 responded to our call for participation in the present study and were recruited. Then, 81 cases (80 males and 1 female) gave consent for blood sampling. The only female patient was excluded from the study to prevent discrepancy in data.

The diagnosis of Buerger’s disease was made using Shionoya criteria,¹⁵ including: (1) history of smoking, (2) the onset of disease below the age of 50, (3) arterial occlusion below the popliteal artery, (4) involvement of the upper extremities or migratory thrombophlebitis, and (5) absence of risk factors for atherosclerosis other than smoking.^5,16 Additionally, any history or evidence of atherosclerosis, any abnormality on angiogram, indicating proximal embolization or atherosclerosis, autoimmune disease, thrombophilia, embolic event, malignancies, viral diseases, and allergies was excluded.

Study protocol

A total of 80 patients with Buerger’s disease were included. Demographic characteristics and clinical symptoms, including clinical signs and symptoms, duration of the disease, and smoking status were carefully recorded. Patients were categorized into mild (migratory thrombophlebitis, cold sensitivity Raynaud’s phenomenon, or skin discoloration), moderate (chronic ulcers, claudication, or burning pain of the feet at night), and severe (pain at rest or gangrene) groups, based on the severity of symptoms. Erythrocyte sedimentation rate (ESR) was measured. Plasma, serum, and PBMC of banked sample were isolated to be used for the blood tests. To measure immunoglobulins (Ig) A, G, and M, as well as C3 and C4 components of the complement system, nephelometry equipment (Binding site model, Minineph, England) was used to record light absorption. This was carried out in accordance with the guidelines of the Kit (Binding site, England). As for the cell phenotype cluster of differentiation 3+ (CD3+), CD4+, CD8+, CD19+, CD45+, and CD16+CD65+, flow cytometry method was performed by conjugated monoclonal antibodies (Roche, Switzerland). The serum level of IgE and 50% complement hemolysis (CH50) was obtained by ELISA method applying human IgE ELISA Kit (ab195216; Abcam, Cambridge, USA) and CH50 ELISA Kit (Biocompare, USA).

Ethical consideration

The present study was approved by Mashhad University of Medical Sciences Institutional Review Board. Written informed consent was obtained from all patients. Also, this study followed the principles outlined in the Declaration of Helsinki.

Statistical analysis

Data were analyzed by SPSS version 22.0 (IBM, USA). Kolmogorov–Smirnov test was used to test the normality of the distribution of the data. One-way ANOVA and its non-parametric equivalent, Kruskal–Wallis test was conducted to evaluate differences in immunologic outcomes among the patient groups (mild, moderate, severe) with the various disease severities (between-group comparison). In addition, ANCOVA was applied to adjust for the possible effects of demographics and smoking-related variables with a significant difference between the patient groups (i.e. age, age at onset of first signs, ESR, cigarette number, and duration of smoking) on these findings (continuous outcomes). A multiple logistic regression model was utilized to examine the association of immunologic responses with the disease severity. Ninety-five percent confidence interval (CI) was reported, and a p-value ≤0.05 was considered statistically significant,

Results

Eighty patients with Buerger’s disease, with the mean age of 42.85 ± 8.39 years, mean cigarette number of 19.73 ± 8.35, and mean smoking time (duration) of 19.08 ± 1.11 years, were studied. Table 1 presents the clinical characteristics of the patients. Based on physical examination and clinical evaluation, 11 (13.75%), 46 (57.50%), and 23 (28.75%) patients had mild, moderate, and severe disease, respectively. As shown in Table 2, these three groups of patients were comparable in terms of smoking time (p = 0.094) and cigarette number (p = 0.620). However, patients with mild symptoms were significantly older (0.031) and had later onset of symptoms (0.049).

Table 1.

The clinical findings of the patients.

Signs and symptoms	Frequency (%)	Signs and symptoms	Frequency (%)
Pain at rest	21 (26.25)	Skin discoloration	37 (46.25)
Paresthesia	57 (71.25)	Skin changes	33 (41.25)
Claudication	59 (73.75)	Pulse abnormality	76 (95.00)
Chronic ulcer	29 (36.25)	Coldness	30 (37.50)
Gangrene	14 (17.50)	Cold intolerance	61 (76.25)
Amputation	38 (47.50)	Migratory thrombophlebitis	5 (6.25)

Table 2.

The comparison of demographic and smoking variables among patients with the different disease severities.

Variables	Disease severity			p-value
Variables	Mild	Moderate	Severe	p-value
Mean age (SD), years	48.36 (9.90)b	43.82 (8.23)a	40.33 (7.24)a	0.031
Mean age at onset (SD), years	40.45 (9.91)b	35.08 (7.18)a	34.14 (7.43)a	0.049
Mean smoking time (SD), years	24.82 (7.49)	18.44 (10.18)	18.00 (8.29)	0.094
Mean cigarette number (SD)	22.27 (9.84)	18.90 (8.40)	19.95 (7.53)	0.620¹

SD: standard deviation.

Mean and standard deviation values followed by different lowercase letters in the same row are significantly different (p < 0.05).

¹Kruskal–Wallis test.

In complete blood count, the mean percentage of polymorphonuclear (PMN) cells, lymphocytes, and monocytes was 67.38 ± 6.44%, 27.97 ± 5.82%, and 4.77 ± 2.04%, respectively. Based on flow cytometry, expression of CD3+, CD4+, and CD8+ molecules was 69.6 ± 5.8%, 58.7 ± 9.3%, and 33.5 ± 8.3%, respectively. On the other hand, expression of CD19+, CD45+, and CD16+CD65+ was 12.4 ± 4.6%, 96.5 ± 4.0%, and 10.8 ± 6.0%. Furthermore, the mean ESR level was 21.16 ± 26.85 mm/h (minimum: 1, maximum: 120).

The mean levels of the complement components and Igs are summarized in Table 3. C3 was above the normal range (>170 mg/dl) in 35 patients (44.30%), C4 was out of the normal range in 18 patients (22.78%; <15 mg/dl in 1 patient and >55 mg/dl in 17 patients), and CH50 was below the normal range (<101 U/ml) in 39 patients (48.75%). Although IgG levels fell within the normal range (700–2100 mg/dl) in the samples of all patients, IgA was out of the normal range in 10 patients (12.66%; <100 mg/dl in one patient and >420 mg/dl in nine patients), IgM was below the normal range (<80 mg/dl) in 34 patients (45.95%), and IgE was above the normal range (≥160 IU/ml) in 31 patients (38.75%).

Table 3.

Immunologic markers in the patients.

Variable	Minimum	Maximum	Mean (SD)
C3 (mg/dl)	73.4	300.3	168.01 (44.72)/70–170
C4 (mg/dl)	12.6	90.4	42.92 (15.89)/15–55
CH50 (U/ml)	0.262	173	89.22 (40.21)/101–300
IgA (mg/dl)	96.7	524	272.45 (96.05)/100–420
IgG (mg/dl)	746.9	1940	1282.89 (256.17)/700–2100
IgM (mg/dl)	28.3	260	97.49 (49.86)/80–320
IgE (IU/ml)	2	501	181.38 (160.24)/<160

SD: standard deviation (followed by the reference range).

Table 4 compares the mean values of immunologic responses as well as the adjusted differences among the three categories of the disease severity. There was a statistically significant difference in C4 (p = 0.023) and ESR (p = 0.002) as continuous variables. Following adjustment for confounding factors, including age, age at onset, smoking time, and cigarette number, considerable differences were still found in the mean values of ESR between the severe group and the other two. In the adjusted model, the difference in the mean ESR values of the mild and moderate groups with the severe group was 24.20% (p < 0.05) and 23.15% (p < 0.05), respectively. Therefore, age, age at onset, smoking time, and cigarette number carried only significant effects on the patients’ ESR values, while C4 values showed no significant difference in the severe group when compared to the other two (p > 0.05).

Table 4.

The immunologic responses (continuous parameters) and adjusted differences among patients with the different disease severities.

Variables	Disease severity			p-value	Difference in mean (95% CI)^a
Variables	Mild (n = 11)	Moderate (n = 46)	Severe (n = 23)	p-value	Comparison between mild and moderate levels	Comparison between mild and severe levels	Comparison between moderate and severe levels
Mean ESR (SD), mm/h	13.22 (11.39)	15.35 (22.46)	37.04 (34.41)	0.002^b 0.002^a	−1.05 (−21.81 to 19.71)	−24.20^* (−46.65 to −1.75)	−23.15^* (−38.89 to −7.41)
Mean lymphocytes (SD), %	28.27 (4.31)	28.45 (6.26)	26.83 (5.69)	0.578^c 0.877^a	0.63 (−4.33 to 5.59)	1.14 (−4.37 to 6.65)	0.51 (−3.43 to 4.45)
Mean monocytes (SD), %	4.91 (2.02)	4.54 (1.96)	5.17 (2.23)	0.469^b 0.215^a	0.42 (−1.24 to 2.09)	−0.53 (−2.38 to 1.32)	−0.95 (−2.27 to 0.37)
Mean PMN (SD), %	66.82 (5.62)	67.23 (6.69)	68.00 (6.56)	0.780^b 0.786^a	−1.46 (−6.92 to 3.99)	−0.80 (−6.87 to 5.26)	0.66 (−3.67 to 5.00)
Mean CD3+ (SD), %	68.91 (5.34)	68.90 (6.16)	71.50 (5.30)	0.231^c 0.244^a	0.21 (−4.88 to 5.31)	−2.53 (−8.19 to 3.13)	−2.74 (−6.79 to 1.30)
Mean CD4+ (SD), %	54.18 (10.97)	59.27 (8.69)	60.00 (9.44)	0.285^b 0.214^a	−5.34 (−13.58 to 2.90)	−6.32 (−15.48 to 2.84)	−0.98 (−7.53 to 5.57)
Mean CD8+ (SD), %	35.00 (8.44)	33.94 (8.53)	31.81 (7.92)	0.517^c 0.570^a	−0.67 (−7.95 to 6.60)	1.83 (−6.26 to 9.91)	2.50 (−3.28 to 8.28)
Mean CD16+CD65+ (SD), %	12.45 (6.53)	11.14 (6.38)	9.14 (4.61)	0.305^b 0.348^a	1.21 (−4.03 to 6.45)	3.19 (−2.64 to 9.01)	1.97 (−2.19 to 6.14)
Mean CD19+ (SD), %	12.18 (4.17)	12.90 (4.46)	11.48 (5.16)	0.547^b 0.646^a	−0.38 (−4.58 to 3.81)	0.89 (−3.78 to 5.56)	1.27 (−2.06 to 4.61)
Mean C3 (SD), mg/dl	179.04 (27.73)	160.08 (41.04)	179.08 (55.87)	0.062^b 0.229^a	24.78 (−13.10 to 62.66)	11.97 (−29.81 to 53.75)	−12.81 (−42.79 to 17.17)
Mean C4 (SD), mg/dl	50.37 (19.90)	38.76 (13.57)	47.87 (16.18)	0.023^b 0.072^a	11.47 (−2.76 to 25.70)	3.57 (−12.12 to 19.27)	−7.90 (−19.16 to 3.37)
Mean CH50 (SD), U/ml	91.05 (48.05)	91.71 (37.82)	83.98 (43.06)	0.804^b 0.760^a	−3.99 (−40.38 to 32.40)	4.56 (−35.43 to 44.54)	8.55 (−19.90 to 37.00)
Mean IgA (SD), mg/dl	265.65 (102.24)	258.40 (101.33)	305.21 (75.52)	0.166^c 0.244^a	0.13 (−79.10 to 79.36)	−41.73 (−129.13 to 45.66)	−41.86 (−104.58 to 20.85)
Mean IgG (SD), mg/dl	1182.02 (224.33)	1255.64 (224.97)	1384.46 (301.95)	0.052^c 0.126^a	−49.02 (−274.19 to 176.14)	−177.32 (−424.59 to 69.95)	−128.30 (−304.54 to 47.94)
Mean IgM (SD), mg/dl	105.88 (55.85)	96.77 (50.78)	100.98 (48.71)	0.801^b 0.912^a	5.50 (−42.90 to 53.90)	−0.35 (−52.98 to 52.28)	−5.85 (−42.87 to 31.17)
Mean IgE (SD), IU/ml	108.42 (133.02)	199.27 (160.73)	195.99 (166.12)	0.228^b 0.668^a	−53.06 (−198.81 to 92.68)	−47.29 (−207.42 to 112.84)	5.77 (−108.15 to 119.70)

CI: confidence interval; ESR: erythrocyte sedimentation rate; SD: standard deviation.

^aANCOVA adjusting for age, age at onset, smoking time, and cigarette number.

^bOne-way ANOVA.

^cKruskal–Wallis test.

*Significant at the 0.05 level.

Table 5 exhibits the proportions of the immunologic outcomes within the normal range. The findings of the multiple logistic regression model revealed that there were significant differences in the dichotomized values of ESR (p = 0.032) and C4 (p = 0.011). Adjustment for covariates did not change this finding for ESR (p < 0.05). However, C4 only differed significantly in patients with severe symptoms compared to the moderate group (p < 0.05). Based on the adjusted model, the odds ratio (OR) of elevated ESR in the patients with mild and moderate symptoms was 18% (95% CI = 0.03–0.99) and 22% (CI = 0.06–0.79), respectively, versus the severe group. The OR of abnormal C4 (elevated or lowered) in patients with moderate disease was 24% (CI = 0.06–0.90) when compared to patients with severe symptoms. Therefore, this analysis demonstrated a marked direct association of elevated ESR and abnormal C4 with the presence of the severe symptoms in patients with Buerger’s disease.

Table 5.

Normal immunologic outcomes (dichotomous parameters) and odds ratio for the mild and moderate levels versus the severe level with 95% confidence intervals.

Variables	Disease severity			p-value^a	OR (95% CI)^b
Variables	Mild (n = 11)	Moderate (n = 46)	Severe (n = 23)	p-value^a	Comparison between mild and severe levels	Comparison between moderate and severe levels
ESR (<15 mm/h)^c, n (%)	7 (15.2)	31 (67.4)	8 (17.4)	0.032	0.18^* (0.03–0.99)	0.22^* (0.06–0.79)
CD4+ (40–60%), n (%)	7 (18.9)	21 (56.8)	9 (24.3)	0.536	0.39 (0.07–2.05)	0.69 (0.22–2.22)
CD8+ (18–30%), n (%)	4 (14.8)	15 (55.6)	8 (29.6)	0.983	1.07 (0.21–5.45)	1.20 (0.38–3.83)
CD16+CD65+ (5–10%), n (%)	3 (10.3)	17 (58.6)	9 (31.0)	0.669	1.16 (0.21–6.49)	0.74 (0.23–2.36)
CD19+ (6.3–20%), n (%)	10 (15.4)	38 (58.5)	17 (26.2)	0.521	0.43 (0.03–5.15)	0.51 (0.11–2.51)
C3 (70–170 mg/dl), n (%)	4 (9.1)	29 (65.9)	11 (25.0)	0.227	3.84 (0.66–22.36)	0.62 (0.19–2.02)
C4 (15–55 mg/dl), n (%)	7 (11.5)	41 (67.2)	13 (21.3)	0.011	0.87 (0.17–4.46)	0.24^* (0.06–0.90)
CH50 (101–300 U/ml), n (%)	5 (12.2)	25 (61.0)	11 (26.8)	0.806	1.21 (0.25–5.79)	0.67 (0.22–2.05)
IgA (100–420 mg/dl), n (%)	10 (14.5)	40 (58.0)	19 (27.5)	0.927	0.22 (0.01–4.09)	0.73 (0.12–4.14)
IgM (80–320 mg/dl), n (%)	5 (12.5)	20 (50.0)	15 (37.5)	0.432	2.06 (0.38–11.04)	2.22 (0.66–7.43)
IgE (<160 IU/ml), n (%)	8 (16.3)	28 (57.1)	13 (26.5)	0.660	0.39 (0.07–2.09)	0.79 (0.26–2.41)

CI: confidence interval; ESR: erythrocyte sedimentation rate; OR: odds ratio.

^aChi-squared test.

^bModel adjusted for age, age at onset, smoking time, and cigarette number.

^cNormal range.

*Significant at the 0.05 level.

Discussion

Recent evidence has highlighted the role of the immune system in the etiology of TAO. Thromboses are frequently associated with moderate, nonspecific inflammatory infiltrate, primarily involving PMN leukocytes, mononuclear cells, and scarce multinuclear giant cells. Very few studies have addressed the immunological alterations in Buerger’s disease. The present study aimed to investigate the relationship between the Buerger’s disease severity and immunological markers, rather than immunologic mechanisms responsible for the etiology of Buerger’s disease.

The etiology of the disease, based on the existing literature, is related to the effects of tobacco on the immune system, interferon reduction, decreased activity in the provision of antigens and neutrophils, decreased immunoglobulin circulation and inhibition of cytokines, which can attenuate the acquired and inherent immunity, and result in susceptibility to infections. Buerger’s disease may induce inflammation and autoimmune responses by increasing the levels of acute free radicals, releasing intracellular antigens, increasing neutrophil count, promoting spontaneous activity of B cells, as well as augmenting T cells’ circulation and activity of CD4+.^17–19 These highlight the contribution of the immune system to the development of Buerger’s disease.

Pulse abnormality, cold intolerance, and claudication were the most common symptoms in this study. In other studies, common symptoms were superficial thrombophlebitis, claudication, ulcer, and coldness.^20–23 The results of this study showed that C3 and C4 were normal in 55.70 and 77.22% of patients, respectively. Moreover, 51.25% had normal CH50 values. Also, elevated ESR and C4 were associated with the presence of severe symptoms. This finding may reflect secondary inflammation, resulting from some conditions such as gangrene and ulcer in severe cases, as a cause of elevated inflammatory biomarkers.

Accumulation of Igs and C3 component in the vessel wall has been found in several autoimmune vascular disorders, such as polyarteritis nodosa,²⁴ temporal arteritis,²⁵ hypersensitivity angiitis,²⁶ Wegner’s granulomatosis,²⁷ and vasculitis complicating rheumatoid arthritis.²⁸ This linear deposition is observed along the length of the internal elastic lamina.²⁹ Recently, Jorge et al.³⁰ reported that ESR, C3, and C4 values were abnormal in a 34-year-old male patient with ulcers in the fingertips and a history of heavy smoking.

Furthermore, in this study, normal values of IgA, IgG, IgE, and IgM were observed in 87.34, 100, 61.25, and 54.05% of patients, respectively. High serum concentrations of circulating immune complexes, including IgG, IgM, and IgA, were demonstrated by Slavov et al.³¹ in TAO cases. The accumulation of IgG, C3, and C4 was observed in the blood vessels of patients with Buerger’s disease.³² Huang et al. described a 34-year-old patient diagnosed with TAO, heavy cigarette smoking, and refractory ulcerations on her ankles. The results indicated that she had a high IgE of 12,500 IU/ml (normal range <165). Thus, omalizumab, as an anti-IgE can be beneficial for the treatment of TAO.³³ In another study, levels of all serum Igs showed a significant rise in TAO patients as compared with controls.³⁴ This finding was also supported by the findings of Gulati et al.³⁵

García et al. reported a significant increase in IgA levels of patients with Buerger’s disease. This finding along with the accumulation of IgA on mesangium and C3 without C1q and C4 illustrates the activation of the complement system, which can explain the role of IgA in the pathogenesis of Buerger’s disease.³⁶ In a study by Roncon de Albuquerque et al.,³⁷ it was found that patients with Buerger’s disease had immune complexes in their blood circulation. However, healthy participants and those with atherosclerosis did not have such complexes.

Zheng et al. investigated the immunological changes in patients with Buerger’s disease using humoral immunity, cellular immunity, and immunopathologic manifestations. In humoral immunity, gamma globulin levels, immune complex, and IgG levels increased. In cellular immunity, T cells and suppressor cells decreased. In their study, infiltration of lymphocyte, neutrophils, monocytes, and immune complex was detected in all involved layers of the vessels. Their preliminary finding introduced Buerger’s disease as an autoimmune disease associated with the antigen–antibody complex.³⁸

To investigate the role of cellular immunity in Buerger’s disease, the phenotypes of the immune cells were examined. CD3+, CD4+, CD8+, CD16+CD65+, and CD19+ were out of the normal range in 13.5, 50, 63.51, 12.16, and 17.57%, respectively. In a study by Kobayashi et al., it has been demonstrated that this disease is mainly associated with cell infiltration seen in the thrombus and intima. Meanwhile, CD3+ T cells are much larger than CD20+ B cells. CD68+ macrophages or S-100 protein-positive dendritic cells are present throughout the acute and subacute stages, especially in the intima. Moreover, IgA, IgG, IgM, and complement factors (C3 and C4) are deposited along the internal elastic lamina.²⁹ The level of cellular infiltration in Buerger’s disease is higher in acute and subacute lesions as compared with chronic lesions. In acute lesions, the number of TCD3+ cells is higher than BCD20+ cells. However, T cells (CD3+) become rarer than B cells (CD20+) in the chronic phase. In acute phase, the number of CD8+ T cells is equal to the number of CD4+ T cells, which is not normal. When the lesion becomes chronic, the number of CD8+ cells becomes higher than that of CD4+ cells.^29,39

Our study was limited by the lack of a control group for comparison. Another limitation was that most of our patients with Buerger’s disease did not respond to our call to participate in this study. In fact, roughly one-third of patients (80 out of 225) were recruited and gave consent for blood sampling. In addition, changes in the intensity and severity of symptoms had occurred since the disease onset in some patients, and not all patients were at the active phase of the disease. On the other hand, medications received by the patients, smoking status, presence of tissue loss (ulcer or gangrene), and associated inflammation could potentially alter the immune response.

Conclusion

In conclusion, the present study indicates that the progression of Buerger’s disease has led to significant alterations in ESR and C4 levels. Changes in these inflammatory biomarkers can be considered a secondary consequence of an inflammatory process, such as ulcer and gangrene in patients with severe disease.

Supplemental Material

VAS910055 Supplemental Material - Supplemental material for Humoral and cellular immune response to Buerger’s disease

Supplemental material, VAS910055 Supplemental Material for Humoral and cellular immune response to Buerger’s disease by Seyed Morteza Ehteshamfar, Jalil Tavakkol Afshari, Mohammad-Hadi S. Modaghegh, Mahmoud Mahmoudi, Gholam Hosein Kazemzadeh and Fatemeh Sadeghipour Kermani in Vascular

Footnotes

Acknowledgments

We would like to thank vice chancellor for research of Mashhad University of Medical Sciences for approving the proposal of this study. We thank Mrs Elham Lotfian, research assistant of Vascular and Endovascular Surgery Research Center, for her kind assistance in preparing the manuscript for submission. This study has been extracted from the result of a PhD thesis, conducted in Vascular and Endovascular Surgery Research Center and Immunology Research Center of Mashhad University of Medical Sciences, Mashhad, Iran.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Mohammad hadi Saeed Modaghegh

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