HANDOVER

Activated neutrophils express adhesion molecules, which enable them to stick to endothelial cells^^^ and degranulate^^^’^^^’^'^’^^^, releasing fi-ee radicals^^^, peroxidases and proteolytic enzymes^^^. Using these products, such as lysozyme and elastase, to study leucocyte activation is limited because they give an indication of past, local events but no information about the activity of leucocytes in the systemic circulation. Interestingly, we found no real difference between controls and claudicants. Both show increase immediately post-exercise following which plasma elastase rapidly retums to baseline. However, when elastase levels are normalised for white cell and neutrophil count, controls show higher levels compared to patients. Furthermore, controls appear to have a biphasic response, with increases immediately post-exercise and at about 35 minutes, whereas, patients show a brief fall at 5 minutes post-exercise, but otherwise little variation. It is possible that the healthy controls produce higher increases in blood flow with exercise and destroy more effete neutrophils. The fall in patient elastase may arise

because of a greater absolute increase in neutrophil count, thus “diluting” the increase in elastase levels. This would suggest ischaemia within claudicating muscle is insufficient to cause degranulation and elastase release. These findings appear at odds with those of the Edinburgh study^^^ and others^^’^^^ which found increased elastase levels in claudicants and the reports of Hickey et al who found increased lysozyme levels in claudicants^"^\ The numbers in our study may have been too small to detect a difference or increased levels reported in the above two studies may have been due to exercise prior to the study period.

3. Neutrophil activation results.

Studies of systemic neutrophil activity have demonstrated decreased filterability^"*^"^^^ following exercise in claudicants compared to controls. Experimental work, using an animal model of claudication, has shown increased neutrophil-endothelial cell adhesion and endothelial cell swelling, both locally and systemically^"^^’^"^^. However, in all these studies the ‘markers’ of activation have been investigated immediately or shortly after exercise. The duration of these effects is unclear and it is possible that activated neutrophils may be ‘filtered out’ after a few passes through the microcirculation. Hence, their capacity to cause increased damage in say MI or stroke may be limited to the immediate post-exercise period.

Our study differs in that it considers both activation and the activation potential in neutrophils before and up to two hours post-exercise. We found no increase in unstimulated neutrophil activity, by either the superoxide (SO) or hydrogen peroxide (H2O2) techniques, following exercise. This differs from the finding of increased

filterability and adhesiveness post-exercise in other studies^'^^’^'^^’^^^. However, direct comparison of the studies is difficult. Firstly, our patients had all rested that morning, the study being their first episode of claudication. This information is not given in other reports, hence patients may have developed several episodes of ischaemia and reperfusion prior to their investigation. Secondly, increased filterability of neutrophils occurs with activation, but activation also causes free radical release, as demonstrated by increases in lipid peroxides^^^’^"^. Hence, such neutrophils maybe depleted of free radicals and not appear activated without further stimulation.

Stimulation of the neutrophils, albeit with a non-physiological stimulant, showed increased release of SO and H2O2 in claudicants compared to controls, the effect lasting

up to 2 hours. The assumption is made that a similar increase would occur in response to physiological stimulation, and under physiological, i.e., in vivo conditions. These findings suggest that following claudication neutrophils are primed, and if further stimulated may produce a more “aggressive” release of free radicals. Cuiffetti et al^^^ also looked at neutrophil SO production following claudication, but only in the immediate post-exercise period. They found a significant fall in neutrophil SO production, and a rise in plasma oxidant capacity indicating free radical release. The implication is that the free radicals are derived from neutrophils, which then become refractory to further stimulation. We also found an initial fall in neutrophil SO production but this rapidly returned to normal. Subjects in their study performed two consecutive exercise tests and this may explain the larger fall they observed. To our knowledge neutrophil H2O2 production, measured using

flow cytometric techniques, has not been reported in claudicants. This technique is considered a more accurate and sensitive measure of neutrophil activation^^^. This is partly because cell preparation is less traumatic and because flow cytometry allows

assessment of individual cells. Using this technique, and the superoxide assay, we have shown an increase in neutrophil activation potential following claudication. This could be partly explained by the increase in numbers of circulating neutrophils, particularly if these extra cells had a greater activation potential. However, both groups showed an increase in neutrophils, and no increase in activation potential was found in the control group. Furthermore, the increase in neutrophil counts is over within 20 minutes, whereas H2O2 peaks at 40 minutes. Claudicants may have a pool of neutrophils with increased

activation potential, but such cells would tend to marginate because of increased adhesive properties. Increased adhesiveness has been reported, and such cells bind firmly to the endothelium^^^ and are not released with increased flow^^^. Furthermore, such ‘sticky’ cells would be expected to return to the marginated pool preferentially to normal cells. Alternatively, the trapping of activated leucocytes, particularly in the reperfusion stage, and the destruction of effete cells, would lead to an increased proportion of the normally marginated leucocytes. If these marginated white cells were comparatively juvenile, due to increased turnover, this may explain the finding of long-term increased activation potential. However, without further data on leucocyte turnover in peripheral vascular disease and the dynamics of the various leucocyte “pools” during exercise, in claudicants and controls, it is difficult to attribute our findings to shifts in leucocyte populations.