Neutrophil activation and mediators of inflammation in chronic venous insufficiency.

Philip D Coleridge Smith DM FRCS, Reader in Surgery

Department of Surgery, University College London Medical School, The Middlesex Hospital, Mortimer Street, London W1N 8AA.

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Summary for Phlebology Digest
Venous ulceration continues to be a problem which eludes simple cure. A considerable amount of work has been done to identify the pathological mechanisms at work in this disease, but the exact sequence of events which leads to leg ulceration has yet to be established. It is well known that ulceration occurs some patients when the muscle pumping mechanisms of the leg are impaired due to disease in the superficial or deep venous systems . The consequence of incompetent lower limb vein valves is that the pumping mechanism no longer reduces the pressure in the veins to low levels during walking leading to damage to the microcirculation of the skin.
In the vascular system, leucocytes are exceptionally important in the pathogenesis of ischaemia. This led to a study of the effects of venous hypertension on leucocyte metabolism. Moyses studied the limbs of normal subjects in response to raised venous pressure, and measured haematological parameters to assess the effect of venous hypertension, showing that this results in leucocyte sequestration in the lower limb. Thomas performed a similar study in which he compared subjects with normal lower limbs to those of patients with venous disease, lipodermatosclerosis and ulceration . His patients were asked to sit with their legs dependent to produce venous hypertension and blood samples were taken from the long saphenous vein at the ankle. After 60 minutes patients with venous disease were 'trapping' 30% of the white cells and control subjects were trapping 5%. I investigated the microcirculation of the skin with a capillary microscope, and combining my observations with those of Thomas, published a hypothesis suggesting that white cell trapping resulted in neutrophil activation, causing damage to the tissues .
Bollinger has investigated the skin microcirculation in venous disease using fluorescence video capillary microscopy . He measured the rate of diffusion of fluorescein out of capillaries after an intravenous injection. He showed that capillaries in venous disease are much more permeable than normal to this molecule, contrary to the suggestions made in the 'fibrin cuff' hypothesis of Browse and Burnand . Using simultaneous fluorescence and light capillary microscopy Franzeck has described the appearances of capillary loops which are filled with red blood cells, but did not appear to be perfused . He suggested that this may be due to capillary 'thrombosis'.
At The Middlesex Hospital, control subjects were exposed to lower limb venous hypertension produced by standing with blood samples taken from the hand and the leg veins. Degranulation of neutrophils was studied by measuring plasma levels of neutrophil elastase (a primary neutrophil granule enzyme) and lactoferrin (a secondary neutrophil granule enzyme). After a 30 minute period of experimental venous hypertension, a rise in plasma lactoferrin concentration was observed in the blood taken from the foot and from the arm . When venous hypertension was produced by inflation of a cuff around one lower limb, a rise in lactoferrin was only observed in that limb. Subsequently expression of the surface neutrophil ligand, CD11b, has been investigated as a marker of neutrophil activation. The experiment was repeated as before on control subjects. Blood was taken from a dorsal foot vein. CD11b expression was assessed by fluorescent labelled monoclonal antibody used to label neutrophils in whole blood which were counted using flow cytometry. During the period of ambulatory venous hypertension in control subjects, no rise in CD11b expression was seen in the lower limb blood . Following return to the supine position, when neutrophils might be expected to leave the lower limb, according to the studies of Thomas Error! Bookmark not defined., increased levels of CD11b were observed. This indicates that neutrophils were upregulated by their period of adhesion to normal endothelium. An increased white cell:red cell ratio was also observed during this phase confirming white cell egress from the lower limb.
In recent studies undertaken in the Department of Surgery, University College London, measurements of plasma levels of endothelial adhesion molecules have been performed along with von Willibrandt factor. These may reflect endothelial injury in the microcirculation. Patients with chronic venous disease (a group with uncomplicated varicose veins and a group with skin changes) were again studied and compared to normal controls. The concentration of soluble VCAM (vascular endothelial adhesion molecule) was elevated in both patient groups compared to control subjects, and was highest in the group with skin changes (fig.). Smaller elevations of von Willibrandt factor, soluble ICAM and E-selectin were also observed. All volunteers were then subjected to venous hypertension produced by standing for 30 minutes. Further rises of soluble adhesion molecules were noted, of which the rise in sVCAM was the most marked, and was greatest in the patients with skin changes attributable to venous disease. These elevations of soluble endothelial adhesion molecules probably reflect endothelial injury in response to short term experimental venous hypertension.

Pharmacological treatment in venous disease
Although bandaging and stockings have been used effectively in the treatment of chronic venous insufficiency for many years modern pharmacological science may provide assistance in healing venous ulcers. Enhancing fibrinolysis has been attempted to promote removal of the fibrin cuff . This particular treatment did not improve ulcer healing. Drugs which reduce white cell activation may be useful in healing venous ulcers. Pentoxifylline (Trental, Hoechst AG, Germany) reduces the likelihood of white cell activation by an effect which appears to be independent of other known activators of neutrophils such as TNF?, resulting in much lower likelihood of endothelial adhesion . In a multi-centre study in which 82 patients were entered, pentoxifylline has been shown to result in much better healing rates of ulcers than placebo . A further randomised, placebo controlled study in 200 patients has now been completed where higher levels of compression were used . Here although a trend towards improved venous ulcer healing was seen in the active treatment group, statistical significance was not reached. Clearly, effective compression has a greater effect on wound healing than does pentoxifylline. However, this drug may still be useful in resistant ulcers .
A recent study has been undertaken in my laboratory to investigate the efficacy of purified micronised flavonoid fraction (MPFF, Servier, France) which is widely used in the management of the symptoms of venous disease. 20 patients with chronic venous disease (CEAP clinical stage 2-4) were included. Blood was collected from a foot vein and plasma soluble vascular cellular adhesion molecule (VCAM), inter-cellular adhesion molecule 1 (ICAM-1), E-selectin, P-selectin, lactoferrin, von Willibrand factor and VEGF levels were determined using a sandwich ELISA method. In addition, neutrophil and monocyte CD11b and L-selectin were assessed by a flow cytometric technique. Patients were treated for 60 days with MPFF taken orally. Further foot vein blood samples were taken within one week of the end of treatment and further assays done. Plasma levels of soluble endothelial adhesion molecules VCAM and ICAM-1 decreased significantly during the treatment period as well as leucocyte L-selectin expression.
Conclusions
The mechanisms which result in venous ulceration remain under investigation. The most effective non-surgical treatment is still compression bandaging, a method which has been in use for thousands of years. Advances in drug treatment appear to offer the next avenue of advance in the treatment of venous disease. I think that the combined use of pharmacological treatment and compression will eventually become commonplace in the management of venous ulceration.

Keywords:
Venous ulceration, neutrophil, monocyte, leukocyte trapping, endothelial adhesion molecules, flavonoid treatment.

Figure

Figure legend.
1. Plasma VCAM-1 levels in normal controls and patients with chronic venous disease (with and without skin changes), before and after venous hypertension. Descriptors: medians and inter-quartile ranges; statistics; Wilcoxon and Mann Whitney U test for unpaired data. L = lying, S = standing.

References

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