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It has been suggested that the marked cellular inflammatory response, which characterises Behçet’s syndrome (BS), may be attributable to increased neutrophil locomotion.1-4However, others disagree.5 6 We have re-evaluated chemotaxis of polymorphonuclear leucocytes (PMNs) in BS among a greater number of patients in a controlled setting.
Fifty four male BS patients, nine male patients with ankylosing spondylitis, eight with psoriasis and 37 male healthy controls were studied with 28 female patients with BS and 16 healthy female controls. Behçet patients with severe disease were those with active major vessel and/or eye involvement.
We measured chemotaxis with the “under the agarose method”.7 8 The measurements were masked with the assessors not knowing the diagnoses. An inverted microscope fitted with an ocular micrometre disc to measure the migration of neutrophils from middle wells to outer (chemotaxis) and inner wells (chemokinesis) was used. Zymosan activated sera (patients or controls) were used as a source of C5a. Results were expressed as micrometre square (1 mm=8 squares). Additionally the plates were evaluated macroscopically for observation of the migration between neutrophil wells.
Tables 1 and 2 show the results. There were no significant differences between the chemotactic indices of the various groups of patients and controls studied of either sex. Maximal chemotaxis rates in the groups varied from 67% to 100%.
The Boyden millipore filter system has extensively been used for chemotaxis experiments.9 10 The agarose method is simple and cheap. This method can preferentially be used to differentiate chemokinesis from chemotactic migration.7
There is marked heterogeneity in disease expression in men and women in BS11 and we reasoned that some of the confusion in the literature about neutrophil activity might be related to this. Thus we analysed our data separately for either sex. Although there was a tendency for male patients with severe disease to have higher chemotactic indices this was not statistically significant (p=0.62). We did not study any diseased controls for female patients with BS.
Abdulla and Lehner6 observed decreased chemotaxis in BS. Fordham et al,2 on the other hand reported increased chemotaxis, but normal random migration. While Wilkinson,5 similar to our experience, observed normal chemotaxis in BS, more recently Carletto et al 3 reported augmented chemotaxis especially in the active phases of the disease. Finally, Ben Ezra et al, among a group of Behçet patients with uveitis could demonstrate increased chemotactic activity only among a few of these patients, compared with that observed among patients with other forms of uveitis. They concluded that increased chemotactic activity was not a regular feature of ocular BS (personal communication).
In vivo assays do not differentiate chemotaxis from chemokinesis. In the Carletto study clinically active Behçet patients demonstrated increased chemotaxis to sera by Senn’s modified in vivo assay.3 Others had found hyperchemotaxis to neutrophil cytoplasmic fractions again by using an in vivo assay.4Although it is difficult to compare the results of in vitro and in vivo assays, we thought these reported increases might have resulted from increased chemokinesis. In our experiments we observed maximal chemotaxis (3 mm) frequently, however we did not find any significant differences in chemotactic indices between diseased and healthy subjects.
An interesting aspect of our study was the migration between neutrophil wells that was observed in many of the Petri dishes. This was observed even though we had not used cellular materials as chemotactic agents. Presumably the gravity of the cellular materials overcame the chemical gradient of zymosan activated sera in some Petri dishes. Because of the observed migration between neutrophil wells, we suggest that there should be only one “triple well rank” in a Petri dish. On the other hand our method of preincubation of the whole blood for 45 minutes at 37°C before harvesting the PMNs (intended for better viability) might have been responsible for this phenomenon by increasing the chemotactic activity in all groups studied. Further studies are needed to clarify these issues.
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