Adherence Test of Blood Immune Cell to Wound Tissue Reveals Subsets Participating in Healing

Background: Circulating polymorphonuclear leucocytes are recruited in the wound by soluble mediators, causing cell adherence, transcapillary migration and chemotaxis. In a later stage T cells play a modulatory role in wound healing, although the full range of their effects is incompletely understood. There is no evidence that B lymphocytes play a significant role in wound healing.  

Aim: The question arises which and when immune cells and their specific subsets accumulate in the fast and delayed healing wounds. This applies particularly to lower limb non-healing ulcers.

Methods: In order to define which blood immune cells reveal predilection for wound cells or matrix, an in vitro adherence test was worked out. Briefly, cryopreserved tissue sections were covered with blood leukocyte suspension at 4C, incubated for 30 min, the non-adherent cells were washed out. This test allowed to show a) adhesion to fibroblasts, keratinocytes, endothelial cells, matrix, b) phenotypes of adhering cells stained with monoclonal antibodies. Venous ulcer edge biopsy specimens obtained from 15 patients and skin fragments of 5 healthy undergoing varicous vein surgery were studied.

Results: Ulcers. Neutrophils adhered to granulation tissue around capillaries and to matrix, forming clusters. Few adhered to fibroblasts. They also stuck to epidermis. CD68+ve and elastase+ve monocytes were evenly distributed close to capillary lumen. Few scattered irregularly distributed CD3 T cells were seen on granulation tissue but none on epidermis. On normal skin section adherence of neutrophils, monocytes and lymphocytes was almost nil. Interestingly, all cell types strongly adhered to glass.

Conclusions: Preponderance of neutrophils and monocytes over lymphocytes on granulation tissue sections suggests presence of a signal for their accumulation as in the scavenging phase of wound healing. The signal might be microbes and autoimmune tissue-antigen-specific cohorts of granulocytes and lymphocytes.