D. A. Shields’s research while affiliated with UCL Eastman Dental Institute and other places

Leukocyte trapping due to leukocyte-endothelial activation has been implicated as the cause of lipodermatosclerosis and ulceration in patients with chronic venous disease. We investigated endothelial activity in normal controls and patients subjected to short-term venous hypertension. Twenty-five normal volunteers and 30 patients with chronic venous disease divided into two groups: varicose veins with skin changes (LDS, n = 15); and varicose veins without skin changes (VVs, n = 15) were studied. Blood samples were taken from a foot vein before and after experimental venous hypertension. Plasma levels of ELAM-1 (endothelial leukocyte adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), and von Willebrand factor (vWf) was measured by an ELISA. There was a significant rise in the plasma concentration of ELAM-1, ICAM-1 and VCAM-1 in patients and normal controls in response to venous hypertension. Basal levels of plasma VCAM-1 and vWf were higher in patients with LDS compared to patients with VVs. The magnitude of rise of VCAM-1 was greater in patients with LDS compared to patients with VVs (p = 0.01, Mann-Whitney U-test). There was no difference in the basal levels or in the magnitude of change in plasma ICAM-1 and ELAM-1 between the two patient groups. Venous hypertension results in endothelial activation which may aid endothelial-leukocyte adhesion. Patients with LDS exhibit increased VCAM-1, which is a counterligand for receptors expressed by monocytes and lymphocytes signifying that these cells may be more important in the development of skin changes.

Objective To investigate leucocyte activation in normal volunteers subjected to short-term venous hypertension by measuring the cell surface expression of the integrin CD11b and the selectin CD62L on neutrophils and monocytes before and after short-term venous hypertension. Plasma levels of L-selectin, which is shed by leucocytes upon activation, were also measured. Design Prospective study, measuring white cell count, neutrophil and monocyte surface CD11b and L-selectin expression in whole blood using a fluorescent-labelled monoclonal antibody in a flow cytometer, and plasma L-selectin by a commercially available ELISA. Setting The Middlesex Hospital Vascular Laboratory, a referral centre for the investigation of venous diseases. Subjects Twenty-five normal volunteers without any history or clinical finding of venous disease. Results There was a significant fall in the white cell: red cell ratio ( p = 0.03, Wilcoxon) in response to venous hypertension, followed by a significant increase ( p < 0.001, Wilcoxon) when the venous hypertension was reversed. Neutrophil and monocyte CD11b levels fell significantly ( p < 0.001 and p < 0.001, Wilcoxon) in response to venous hypertension. On reversing the venous hypertension a significant increase of CD11b expression was noted in both neutrophils and monocytes ( p < 0.001 and p < 0.01, Wilcoxon). Surface L-selectin expression, however, showed a significant sustained fall in both neutrophils and monocytes (neutrophils, p = 0.05 and p = 0.02; monocytes, p = 0.04 and p = n.s., Wilcoxon) throughout the experiment. There was a significant rise in soluble L-selectin in the plasma following venous hypertension ( p < 0.001, Wilcoxon). Conclusion This study demonstrates both neutrophil and monocyte activation in the microcirculation of normal volunteers in response to short-term venous hypertension.

It has been suggested that leukocyte trapping and activation in the microcirculation of the leg skin causes lipodermatosclerosis and ulceration in patients with chronic venous disease. Ambulatory venous hypertension is accepted as the physiologic factor that leads to ulceration. We investigated leukocyte endothelial adhesion in patients who were subjected to short-term venous hypertension. Two groups of patients with venous disease were studied: group 1, varicose veins with skin changes (n = 15); and group 2, varicose veins without skin changes (n = 15). Blood samples were taken from a foot vein before and after standing for 30 minutes to raise the venous pressure in the lower limb, and after lying supine again for 10 minutes. The samples were analyzed for leukocyte surface CD11b and L-selectin (CD62L) expression using a flow cytometer. Plasma-soluble L-selectin was also measured using an enzyme-linked immunosorbent assay. In patients with skin changes, median neutrophil CD11b levels fell from 4.66 to 3.83 arbitrary units (p = 0.005, Wilcoxon) after 30 minutes of venous hypertension, Median monocyte CD11b levels fell from 7.65 to 5.8 arbitrary units (p = NS, Wilcoxon) after venous hypertension and then fell further to 5.43 arbitrary units (p = 0.02 vs baseline; Wilcoxon) when the venous hypertension was removed. Neutrophil and monocyte L-selectin levels also fell in response to venous hypertension, remaining low even after venous hypertension was removed. A similar pattern was seen in patients with uncomplicated varicose veins. There was a rise in soluble L-selectin in the plasma of both groups of patients after venous hypertension, reflecting leukocyte adhesion to endothelium. In the group of patients with skin changes the level of soluble L-selectin rose from 695 ng/ml to 836 ng/ml (p = 0.02, Wilcoxon), and in the group without skin changes the rise was from 700 ng/ml to 801 ng/ml (p = 0.02, Wilcoxon). Venous hypertension results in sequestration of the more activated population of neutrophils and monocytes in the microcirculation of the leg in patients with venous disease. These cells bind to the endothelium, releasing L-selectin, and do not emerge from the limb when venous hypertension is reversed. These findings do not differ between patients with varicose veins and those with skin changes.

Objective To measure soluble CD54 (ICAM-1) and CD62E (E-selectin) as markers of neutrophil adhesion in four groups of patients with varying severity of venous disease and compare the values obtained with those in age- and sex-matched control subjects. Design Prospective study of patients with varicose veins compared with a group of control subjects with no history or clinical findings of varicose veins. Setting The Middlesex Hospital Vascular Laboratory, London. Patients Patients referred to the Middlesex Hospital Vascular Laboratory for investigation of venous disease. Neither patients nor controls had arterial disease, any other systemic illness, or were on any medication known to alter white cell activity. Interventions Ten millimetres of blood taken from an arm vein into EDTA for a neutrophil count and soluble CD54 and CD62E, measured using an ELISA. Results Similar levels of soluble CD54 and CD62E were found in all four groups of patients compared with their controls ( p = 0.71 for soluble CD54 for all patients compared with all controls, and p = 0.65 for soluble CD62E, Mann–Whitney U-test). There was no difference in the neutrophil count between the controls and patients in any group ( p = 0.74 for all subjects, Mann–Whitney U-test). Conclusion This study shows no evidence of increased soluble CD54 or CD2E or CD62E in patients with venous disease, despite previous work showing increased CD54 and neutrophil degranulation in patients with venous disease. The reason for this is currently unknown.

Leukocyte trapping and activation in the microcirculation of the legs may play an important role in causing skin damage in venous disease. Leukocyte emigration from the microcirculation and subsequent locomotion in response to venous hypertension was studied in a group of 12 normal volunteers using a "skin window" technique. Two 0.5-cm square dermal abrasions were made with a dental stone over the gaiter area of the leg and the flexor aspect of the forearm (control), which were covered with moist micropore membranes. The volunteers lay supine for 30 minutes, and then stood supported for 30 minutes to raise the venous pressure in the leg, and then lay supine again for 30 minutes. The experiment was repeated in six volunteers who lay supine for the whole period. The membranes were changed and collected every 15 minutes, fixed in formal saline solution, and dual-stained for monocytes and polymorphonuclear leukocytes. The type and numbers of cells that emigrated and the furthest distance traveled (leading front) by the cells through the membrane were measured. Both in arms and legs, the vast majority of cells that emigrated were neutrophils, with very few monocytes (arm, 93% neutrophils and 7% monocytes; leg, 97% neutrophils and 3% monocytes). In the 30 minutes after venous hypertension, leukocyte migration significantly decreased in the leg (median leukocyte locomotion: basal, 75.3 microns; standing, 73.5 microns; after hypertension, 62.9 microns; p = 0.012, Wilcoxon matched pairs signed rank test), but not in the arm (basal, 86.2 microns; standing, 84.4 microns; after hypertension, 85.5 microns; p = NS) or when the experiment was repeated with the volunteers lying supine for the entire period (basal, 91.5 microns; standing, 89.4 microns; after hypertension, 92.6 microns; p = NS). Leukocyte migration is decreased immediately after experimental venous hypertension, which may be a result of the release of factors that inhibit migration.

Objective: To determine whether neutrophil CD11b, a marker of neutrophil adhesion, differs in patients with varying degrees of severity of venous disease, and to compare the values obtained with those of age-matched normal control subjects. Design: Prospective study, measuring white cell count and neutrophil CD11b expression in whole blood using a fluorescent-labelled monoclonal antibody in a flow cytometer. Setting: The Middlesex Hospital Vascular Laboratory, a referral centre for the investigation of venous disease. Patients: Ten patients with uncomplicated varicose veins, 10 patients with skin changes of lipodermatosclerosis (LDS), and 20 age-matched control subjects with no history or clinical finding of venous disease. Results: Higher levels of CD11b were found in patients with uncomplicated varicose veins compared with their controls (median 4.6 cf. 1.43 for normal controls, p = 0.005, Mann-Whitney U-test, difference between medians 2.7, 95% confidence interval 1 to 4.6), and lower levels in patients with LDS (median 1.22 cf. 1.53 for normal controls, p = 0.028, Mann-Whitney U-test, difference between medians 0.45, 95% confidence interval 0.02 to 1.3). There was no difference in the white cell or neutrophil count between the patient and control groups. Conclusions: This study demonstrates increased neutrophil surface CD11b expression in patients with uncomplicated varicose veins, but decreased levels in patients with LDS. This might be due to up-regulation of CD11b in some neutrophils with subsequent adhesion, so that only those with low expression remained in the peripheral circulation. Alternatively, this might represent either down-regulation or chronic exhaustion of neutrophil CD11b in these patients.

The white cell trapping hypothesis suggests that raised venous pressure causes white cell margination and activation, with the release of proteolytic enzymes and Superoxide radicals, believed responsible for the tissue destruction seen in chronic venous insufficiency [1].

The white cell trapping hypothesis suggests that raised venous pressure causes white cell margination and activation, with the release of proteolytic enzymes and Superoxide radicals, believed responsible for the tissue destruction seen in chronic venous insufficiency [1].

The plasma elastase level was measured as a marker of neutrophil degranulation in three groups, each of 15 patients, with uncomplicated varicose veins, lipodermatosclerosis (LDS) and venous ulceration. The values obtained were compared with those in age- and sex-matched control subjects. Significantly higher levels of elastase were found in all patient groups compared with controls: median 25.6 ng/ml for patients with uncomplicated varicose veins, 22.1 ng/ml for those with LDS, 26.0 ng/ml for those with venous ulceration. There was no difference in neutrophil count between the patient and control groups. These results provide evidence of increased neutrophil degranulation in patients with venous disease. The finding of raised elastase levels in all three patient groups shows that this was not due solely to the inflammatory process characterizing LDS and venous ulceration.

Objective To investigate the white cell trapping hypothesis of venous ulceration by measuring plasma lactoferrin as a marker of neutrophil degranulation in normal volunteers in two experimental models of venous hypertension. Design A prospective study of volunteers with no history or clinical evidence of venous disease. Setting The Middlesex Hospital Vascular Laboratory, Mortimer Street, London WIN 8AA, UK. Patients Volunteers within the Middlesex Hospital Vascular Laboratory with no history or clinical findings of venous or arterial disease, no other systemic disease, on no medication known to alter white cell activity, and with no recent infection. Interventions Venous blood was taken from cannulae in both feet and the right arm for a neutrophil count and Plasma lactoferrin, measured using an ELISA, during application of a tourniquet to 80 mmHG for 30 min to the right leg while supine, 5 min after release of tourniquet, and then during a 30 min period of standing. Results During application of a tourniquet to the right leg there was a significant rise in plasma lactoferrin and in lactoferrin corrected for the neutrophil count ( p < 0.05, Wilcoxon). In the unoccluded leg, although Plasma lactoferrin rose, this was not significant when corrected for the rise in neutrophil count. After standing for 30 min, the lactoferrin and neutrophil count increased in all three limbs; corrected lactoferrin showed a significant increase in the legs ( p < 0.02), though not in the arm. Conclusion Increased neutrolphil degranulation occurs during periods of short-term venous hypertension in normal volunteers, in keeping with the white cell trapping hypothesis.