Background The etiology of Achilles tendinopathy still remains unclear. Limited data exists on whether immune-competent cells are present in the tendon.
Objectives To evaluate the presence of T and B lymphocytes, NK cells and macrophages in chronic Achilles tendinopathy using immunohistochemistry and unbiased stereological technique for quantification. Further, we wanted to correlate the amount of each cell type to disease duration as well as tendon thickness and vascularity evaluated by ultrasound immediately before biopsy.
Methods 56 non-ruptured Achilles tendons from 25 women and 31 men with chronic tendinopathy were examined with Doppler ultrasound regarding tendon thickness and vascularity. 17 patients had received local steroid injection or other anti-inflammatory treatment within 6 months before the examination. From all tendons, a biopsy was obtained and stained with hematoxylin/eosin, Van Gieson, toluidin blue, Pearls Blue and NaSDCl in addition to the following immunohistochemical markers: CD2, CD3, CD4, CD7, CD8, CD20, CD34, CD56, CD68(KP1), CD68(PG-M1) and Granzyme-B. The area fraction count (AFC) of positive cells in each sample was determined by quantifying and calibrating the area fraction of positive cells to a standard area in 4 mm thick slides using unbiased stereological techniques. The Mann-Whitney rank sum test and the Spearman correlation test was used (p<0.05 was considered significant).
Results The median age of the patients was 50 years (range 33-69) and the median disease duration was 14 months (range 4-360).
In 56 Achilles tendon biopsies, we found presence of CD2+ cells in 44 (79%), CD3+ cells in 49 (88%), CD4+ cells in 47 (84%), CD7+ cells in 38 (68%), CD8+ cells in 32 (57%), CD20+ cells in 10 (18%), CD34+ cells in 54 (96%), CD56+ cells in 31 (55%), CD68KP1+ cells in 54 (96%), CD68PG-M1+ cells in 49 (88%), Granzyme-B+ cells in 4 (7%), iron positive cells in 13 (23%) and NaSDCl positive cells in 45 (80%).
The T lymphocyte AFC correlated positively to
A: B lymphocyte AFC (CD20∼CD2, CD4, CD7; p<0.01, r-value (0.34-0.44)).
B: Macrophage AFC (CD68∼CD2, CD3, CD4, CD7, CD8; p<0.01, r-value (0.33-0.43)) and the presence of haemosiderophages (iron∼CD2, CD3, CD4, CD7; p<0.02, r-value (0.30-0.38)).
C: NK cell AFC (CD56∼CD2, CD7, CD8; p<0.01, r-value (0.33-0.38)).
Disease duration correlated to B lymphocyte (CD20) AFC (p=0.02, r-value=0.31) and NK cell (CD56) AFC (p=0.01, r-value=0.35). In addition, both B lymphocyte and NK cell AFC were significant higher in patients with a disease duration of more than 14 months compared to patients withh a shorter disease duration (p<0.04). Disease duration did not correlate to either tendon thickness (p=0.70, r-value=-0.07) or Doppler vascularity (p=0.13, r-value=-0.22).
No correlation was found between tendon thickness, Doppler vascularity and any AFC.
Conclusions In 56 biopsies from non-ruptured chronic tendinopathic Achilles tendons, we found a combined presence of macrophages, T and B lymphocytes and NK cells with no signs of granulocytic cellular infiltrate. Longer disease duration was associated with higher area fraction count of B lymphocytes and NK cells. These findings support the theory of chronic tendinopathy being driven by an immunologic process.
Disclosure of Interest None Declared
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