Original Article

Vascular
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Humoral and cellular immune response ! The Author(s) 2020
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DOI: 10.1177/1708538120910055
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Seyed Morteza Ehteshamfar1, Jalil Tavakkol Afshari1,2,

Mohammad-Hadi S. Modaghegh2 , Mahmoud Mahmoudi1,
Gholam Hosein Kazemzadeh2 and
Fatemeh Sadeghipour Kermani3

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 pro-
gressive inflammation and clot formation.1,3 This dis-
ease 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 treat-
ment 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
1
Immunology Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran
2
Vascular and Endovascular Surgery Research Center, Mashhad
University of Medical Sciences, Mashhad, Iran
3
Vascular and Endovascular Surgery Research Center, Mashhad
University of Medical Science, Mashhad, Iran
Corresponding authors:
Mohammad Hadi Saeed Modaghegh, Vascular and Endovascular Surgery
Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Email: modagheghmh@mums.ac.ir; modaghegh@hotmail.com
Jalil Tavakkol Afshari, Immunology Research Center, Mashhad University
of Medical Sciences, Mashhad, Iran.
Email: TavakolAJ@mums.ac.ir
2 Vascular 0(0)
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 occur-
rence 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 cellu-
lar 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 humor-
al 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 circu-
lating immunoglobulins, and inhibition of cytokines; all
of which reduce the acquired and inherent immunity and
lead to susceptibility to infections. These findings indi-
cate 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 discrep-
ancy 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 ath-
erosclerosis, 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 includ-
ed. 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 thrombo-
phlebitis, cold sensitivity Raynaud’s phenomenon, or
skin discoloration), moderate (chronic ulcers, claudica-
tion, 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, nephelome-
try 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 3þ (CD3þ), CD4þ, CD8þ, CD19þ,
CD45þ, and CD16þCD65þ, 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 normal-
ity 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
Ehteshamfar et al. 3

first signs, ESR, cigarette number, and duration of and 10.8
6.0%. Furthermore, the mean ESR level
smoking) on these findings (continuous outcomes). A was 21.16
26.85 mm/h (minimum: 1, maximum: 120).
multiple logistic regression model was utilized to exam- The mean levels of the complement components and
ine the association of immunologic responses with the Igs are summarized in Table 3. C3 was above the
disease severity. Ninety-five percent confidence interval normal range (>170 mg/dl) in 35 patients (44.30%),
(CI) was reported, and a p-value 0.05 was considered C4 was out of the normal range in 18 patients
statistically significant, (22.78%; <15 mg/dl in 1 patient and >55 mg/dl in
17 patients), and CH50 was below the normal range
Results (<101 U/ml) in 39 patients (48.75%). Although IgG
levels fell within the normal range (700–2100 mg/dl)
Eighty patients with Buerger’s disease, with the mean age in the samples of all patients, IgA was out of the
of 42.85
8.39 years, mean cigarette number of 19.73
normal range in 10 patients (12.66%; <100 mg/dl in
8.35, and mean smoking time (duration) of 19.08
one patient and >420 mg/dl in nine patients), IgM
1.11 years, were studied. Table 1 presents the clinical was below the normal range (<80 mg/dl) in 34 patients
characteristics of the patients. Based on physical exami- (45.95%), and IgE was above the normal range
nation and clinical evaluation, 11 (13.75%), 46 (57.50%), (160 IU/ml) in 31 patients (38.75%).
and 23 (28.75%) patients had mild, moderate, and severe Table 4 compares the mean values of immunologic
disease, respectively. As shown in Table 2, these three responses as well as the adjusted differences among the
groups of patients were comparable in terms of smoking
time (p ¼ 0.094) and cigarette number (p ¼ 0.620). Table 3. Immunologic markers in the patients.
However, patients with mild symptoms were significantly
older (0.031) and had later onset of symptoms (0.049). Variable Minimum Maximum Mean (SD)
In complete blood count, the mean percentage of C3 (mg/dl) 73.4 300.3 168.01 (44.72)/70–170
polymorphonuclear (PMN) cells, lymphocytes, and C4 (mg/dl) 12.6 90.4 42.92 (15.89)/15–55
monocytes was 67.38
6.44%, 27.97
5.82%, and CH50 (U/ml) 0.262 173 89.22 (40.21)/101–300
4.77
2.04%, respectively. Based on flow cytometry, IgA (mg/dl) 96.7 524 272.45 (96.05)/100–420
expression of CD3þ, CD4þ, and CD8þ molecules was IgG (mg/dl) 746.9 1940 1282.89 (256.17)/700–2100
69.6
5.8%, 58.7
9.3%, and 33.5
8.3%, respectively. IgM (mg/dl) 28.3 260 97.49 (49.86)/80–320
IgE (IU/ml) 2 501 181.38 (160.24)/<160
On the other hand, expression of CD19þ, CD45þ,
and CD16þCD65þ was 12.4
4.6%, 96.5
4.0%, SD: standard deviation (followed by the reference range).

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.

Disease severity

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).
1
Kruskal–Wallis test.
 4

Table 4. The immunologic responses (continuous parameters) and adjusted differences among patients with the different disease severities.
Disease severity Difference in mean (95% CI)a

Mild Moderate Severe Comparison between mild Comparison between mild Comparison between
Variables (n ¼ 11) (n ¼ 46) (n ¼ 23) p-value and moderate levels and severe levels moderate and severe levels

Mean ESR (SD), mm/h 13.22 (11.39) 15.35 (22.46) 37.04 (34.41) 0.002b 0.002a 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) 0.578c 0.877a 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) 0.469b 0.215a 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) 0.780b 0.786a 1.46 (6.92 to 3.99) 0.80 (6.87 to 5.26) 0.66 (3.67 to 5.00)
Mean CD3þ (SD), % 68.91 (5.34) 68.90 (6.16) 71.50 (5.30) 0.231c 0.244a 0.21 (4.88 to 5.31) 2.53 (8.19 to 3.13) 2.74 (6.79 to 1.30)
Mean CD4þ (SD), % 54.18 (10.97) 59.27 (8.69) 60.00 (9.44) 0.285b 0.214a 5.34 (13.58 to 2.90) 6.32 (15.48 to 2.84) 0.98 (7.53 to 5.57)
Mean CD8þ (SD), % 35.00 (8.44) 33.94 (8.53) 31.81 (7.92) 0.517c 0.570a 0.67 (7.95 to 6.60) 1.83 (6.26 to 9.91) 2.50 (3.28 to 8.28)
Mean CD16 12.45 (6.53) 11.14 (6.38) 9.14 (4.61) 0.305b 0.348a 1.21 (4.03 to 6.45) 3.19 (2.64 to 9.01) 1.97 (2.19 to 6.14)
þCD65þ (SD), %
Mean CD19þ (SD), % 12.18 (4.17) 12.90 (4.46) 11.48 (5.16) 0.547b 0.646a 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) 0.062b 0.229a 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) 0.023b 0.072a 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) 0.804b 0.760a 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) 0.166c 0.244a 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) 0.052c 0.126a 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) 0.801b 0.912a 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) 0.228b 0.668a 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.
a
ANCOVA adjusting for age, age at onset, smoking time, and cigarette number.
b
One-way ANOVA.
c
Kruskal–Wallis test.
*Significant at the 0.05 level.
Vascular 0(0)
Ehteshamfar et al. 5

three categories of the disease severity. There was a
statistically significant difference in C4 (p ¼ 0.023)
and ESR (p ¼ 0.002) as continuous variables.
Following adjustment for confounding factors, includ-
ing 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 car-
ried 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 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 ¼ 0.032) and C4 (p ¼ 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 mod-
erate 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 ¼ 0.03–0.99) and 22% (CI ¼ 0.06–0.79), respective-
ly, versus the severe group. The OR of abnormal C4
(elevated or lowered) in patients with moderate disease
was 24% (CI ¼ 0.06–0.90) when compared to patients
with severe symptoms. Therefore, this analysis demon-
strated a marked direct association of elevated ESR
and abnormal C4 with the presence of the severe symp-
toms in patients with Buerger’s disease.
Discussion
Recent evidence has highlighted the role of the immune
system in the etiology of TAO. Thromboses are fre-
quently associated with moderate, nonspecific inflam-
matory 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 activi-
ty 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 infec-
tions. 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 CD4þ.17–19 These highlight the contribution

Table 5. Normal immunologic outcomes (dichotomous parameters) and odds ratio for the mild and moderate levels versus the
severe level with 95% confidence intervals.

Disease severity OR (95% CI)b

Comparison between
Mild Moderate Severe Comparison between moderate and
Variables (n ¼ 11) (n ¼ 46) (n ¼ 23) p-valuea mild and severe levels 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)
CD4þ (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)
CD8þ (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)
CD16þCD65þ (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)
CD19þ (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.
a
Chi-squared test.
b
Model adjusted for age, age at onset, smoking time, and cigarette number.
c
Normal range.
*Significant at the 0.05 level.
6 Vascular 0(0)
of the immune system to the development of Buerger’s
disease.
Pulse abnormality, cold intolerance, and claudica-
tion were the most common symptoms in this study.
In other studies, common symptoms were superficial
thrombophlebitis, claudication, ulcer, and cold-
ness.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 granu-
lomatosis,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 con-
centrations 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 ciga-
rette 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
Garcıa 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 partici-
pants and those with atherosclerosis did not have
such complexes.
Zheng et al. investigated the immunological changes
in patients with Buerger’s disease using humoral immu-
nity, 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 pre-
liminary finding introduced Buerger’s disease as an
autoimmune disease associated with the antigen–anti-
body complex.38
To investigate the role of cellular immunity in
Buerger’s disease, the phenotypes of the immune cells
were examined. CD3þ, CD4þ, CD8þ,
CD16þCD65þ, and CD19þ were out of the normal
range in 13.5, 50, 63.51, 12.16, and 17.57%, respective-
ly. In a study by Kobayashi et al., it has been demon-
strated that this disease is mainly associated with cell
infiltration seen in the thrombus and intima.
Meanwhile, CD3þ T cells are much larger than
CD20þ B cells. CD68þ 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 com-
pared with chronic lesions. In acute lesions, the number
of TCD3þ cells is higher than BCD20þ cells. However,
T cells (CD3þ) become rarer than B cells (CD20þ) in
the chronic phase. In acute phase, the number of
CD8þ T cells is equal to the number of CD4þ
T cells, which is not normal. When the lesion becomes
chronic, the number of CD8þ cells becomes higher
than that of CD4þ 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 pro-
gression of Buerger’s disease has led to significant alter-
ations in ESR and C4 levels. Changes in these
inflammatory biomarkers can be considered a second-
ary consequence of an inflammatory process, such as
ulcer and gangrene in patients with severe disease.
Ehteshamfar et al.
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 https://orcid.org/
0000-0002-2724-7463
References
1. Klein-Weigel P, Volz T, Gutsche-Petrak B, et al.
Immunoadsorption in Buerger’s disease (thromboangiitis
obliterans): a promising therapeutic option: results of a
consecutive patient cohort treated in clinical routine care.
Lupus Open Access 2016; 1: 2.
2. Narváez J, Garcıa-G 
omez C, Alvarez L, et al. Efficacy of
bosentan in patients with refractory thromboangiitis
obliterans (Buerger disease): a case series and review of
the literature. Medicine 2016; 95: e5511.
3. Rivera-Chavarrıa IJ and Brenes-Gutierrez JD.
Thromboangiitis obliterans (Buerger’s disease). Ann
Med Surg 2016; 7: 79–82.
4. Jimenez-Ruiz C, Dale L, Mochales JA, et al. Smoking
characteristics and cessation in patients with thromboan-
giitis obliterans. Monaldi Arch Chest Dis 2006; 65: 217–
221.
5. Igari K, Inoue Y and Iwai T. The epidemiologic and
clinical findings of patients with Buerger disease. Ann
Vasc Surg 2016; 30: 263–269.
6. Higashi Y, Azuma N, Takeishi Y, et al. Effect of a low-
intensity pulsed ultrasound device, SX-1001, on clinical
symptoms in Buerger disease with limb ischemia. Int
Heart J 2015; 56: 632–638.
7. Kaskurthy S, Gautam A, Ghimire S, et al. Cannabis
use: increasing risk of thromboangiitis obliterans
(Buerger’s disease). World Journal of Pharmacy and
Pharmaceutical Sciences 2015; 4: 1582–1588.
8. De JH, Florez A, Fernandez JL, et al. Treatment of
Buerger disease (thromboangiitis obliterans) with
7
Bosentan: a case report. BMJ Case Reports 2009; 2009:
bcr0820080691.
9. Decousus H, Quere I, Presles E, et al. Superficial venous
thrombosis and venous thromboembolism: a large, pro-
spective epidemiologic study. Ann Intern Med 2010; 152:
218–224.
10. Arnson Y, Shoenfeld Y and Amital H. Effects of tobacco
smoke on immunity, inflammation and autoimmunity.
J Autoimmun 2010; 34: J258–J265.
11. Smolen JS, Youngchaiyud U, Weidinger P, et al.
Autoimmunological aspects of thromboangiitis obliter-
ans (Buerger’s disease). Clin Immunol Immunopathol
1978; 11: 168–177.
12. Kopan R and Ilagan M. The canonical notch signaling
pathway: unfolding the activation mechanism. Cell 2009;
137: 216–233.
13. Suchting S, Freitas C, Le Noble F, et al. The notch ligand
delta-like 4 negatively regulates endothelial tip cell for-
mation and vessel branching. Proc Natl Acad Sci USA
2007; 104: 3225–3230.
14. Cacione DG and Moreno DH. Stem cell therapy for
treatment of thromboangiitis obliterans (Buerger’s dis-
ease). Cochrane Database Syst Rev 2017: 9.
15. Kroger K. Buerger’s disease: what has the last decade
taught us? Eur J Intern Med 2006; 17: 227–234.
16. Dargon PT and Landry GJ. Buerger’s disease. Ann Vasc
Surg 2012; 26: 871–880.
17. Quintero OL, Rojas-Villarraga A, Mantilla RD, et al.
Autoimmune diseases in the intensive care unit. An
update. Autoimmun Rev 2013; 12: 380–395.
18. McKenzie BS, Kastelein RA and Cua DJ. Understanding
the IL-23–IL-17 immune pathway. Trends Immunol 2006;
27: 17–23.
19. Marzouni HZ, Tarkhan F, Aidun A, et al. Cytotoxic
effects of coated gold nanoparticles on PC12 cancer
cell. Galen Med J 2018; 7: 1110.
20. Sasaki S, Sakuma M, Kunihara T, et al. Distribution of
arterial involvement in thromboangiitis obliterans
(Buerger’s disease): results of a study conducted by the
Intractable Vasculitis Syndromes Research Group in
Japan. Surg Today 2000; 30: 600–605.
21. Salimi J, Tavakkoli H, Salimzadeh A, et al. Clinical char-
acteristics of Buerger’s disease in Iran. J Coll Physicians
Surg Pak 2008; 18: 502–505.
22. Modaghegh M-H, Kazemzadeh GH, Ravari H, et al.
Buerger’s disease in the northeast of Iran: epidemiology
and clinical features. Vascular 2015; 23: 519–524.
23. Barlas S, Elmaci T, Dayioglu E, et al. Has the clinical
definition of thromboangiitis obliterans changed indeed?
Int J Angiol 1997; 6: 49–55.
24. Mellors RC and Ortega LG. Analytical pathology. III.
New observations on the pathogenesis of glomerulone-
phritis, lipid nephrosis, periarteritis nodosa, and second-
ary amyloidosis in man. Am J Pathol 1956; 32: 455–499.
25. Liang GC, Simkin PA and Mannik M. Immunoglobulins
in temporal arteries: an immunofluorescent study. Ann
Intern Med 1974; 81: 19–24.
26. Braverman IM and Yen A. Demonstration of immune
complexes in spontaneous and histamine-induced lesions
8 Vascular 0(0)
and in normal skin of patients with leukocytoclastic
angiitis. J Invest Dermatol 1975; 64: 105–112.
27. Fauci AS and Wolff SM. Letter: immunological features
of Wegener’s granulomatosis. Lancet 1974; 1: 688–689.
28. Conn DL, McDuffie FC and Dyck PJ.
Immunopathologic study of sural nerves in rheumatoid
arthritis. Arthritis Rheum 1972; 15: 135–143.
29. Kobayashi M, Ito M, Nakagawa A, et al.
Immunohistochemical analysis of arterial wall cellular
infiltration in Buerger’s disease (endarteritis obliterans).
J Vasc Surg 1999; 29: 451–458.
30. Jorge VC, Ara ujo AC, Noronha C, et al. Buerger’s dis-
ease (thromboangiitis obliterans): a diagnostic challenge.
BMJ Case Rep 2011; 2011: bcr0820114621.
31. Slavov E, Stanilova S, Petkov D, et al. Cytokine produc-
tion in thromboangiitis obliterans patients: new evidence
for an immune-mediated inflammatory disorder. Clin
Exp Rheumatol 2005; 23: 219–226.
32. Roncon de Albuquerque R, Delgado L, Correia P, et al.
Circulating immune complexes in Buerger’s disease.
Endarteritis obliterans in young men. J Cardiovasc Surg
1989; 30: 821–825.
33. Huang Z-H, Kuo S-Y, Chiu Y-H, et al. Treatment of
multiple refractory ankle ulcerations in thromboangiitis
obliterans: a case report. Medicine (Baltimore) 2018; 97:
e10798.
34. Ehrenfeld M, Papa M and Adar R. Autoimmune
mechanisms in Buerger disease. Crit Ischemia 2001; 11:
7–13.
35. Gulati SM, Singh KS, Thusoo TK, et al. Immunological
studies in thromboangiitis obliterans (Buerger’s disease).
J Surg Res 1979; 27: 287–293.
36. Garcıa LG, Gimenez AL, Camacho JD, et al. Berger’s
disease. Study of eleven cases (author’s transl). An Esp
Pediatr 1982; 16: 210–218.
37. Roncon de Albuquerque R, Delgado L, Correia P, et al.
Circulating immune complexes in Buerger’s disease.
Endarteritis obliterans in young men. J Cardiovasc Surg
1989; 30: 821–825.
38. Zheng P, Fu P, Wang W, et al. Immunological studies on
thromboangiitis obliterans. Chin Med J 1989; 102:
129–136.
39. Olin JW. Thromboangiitis obliterans (Buerger’s disease).
N Engl J Med 2000; 343: 864–869.