Migration of neutrophils in patients with paroxysmal nocturnal haemoglobinuria (PNH) was studied using two different complement-free in vitro model systems, subagarose and transendothelial migration. In the subagarose migration assay the mean migration distance of PNH neutrophils was slightly, but significantly, reduced to 1236 microns (range 753-1586, n = 6) compared to a normal mean of 1476 microns (range 1076-1768, n = 6, P = 0.016). By immunocytochemical staining for the urokinase type plasminogen activator receptor (uPAR) which is a glycosyl-phosphatidyl-inositol (GPI) anchored protein expressed by normal, but not by PNH-affected, neutrophils, it was shown that the uPAR-positive subpopulation of normal neutrophils predominated among the faster migrating cells (60-80% normal cells at the front of migration) while uPAR-negative (i.e. PNH-affected neutrophils) were more numerous close to the application well (5-30% normal cells). When migration of neutrophils was tested across a monolayer of human umbilical vein endothelial cells (HUVEC) cultured on polycarbonate filters, there was a 3-4-fold impairment of the migration of the PNH-affected neutrophils both in the absence of stimulation and after stimulation with fMLP (P < 0.001 in both cases). After IL-1 stimulation of the endothelium the impairment was even more pronounced (8-fold difference, P < 0.001). When the endothelial cells were grown on collagen-coated filters the impairment of the migration of PNH neutrophils was less pronounced, but still significant after stimulation with fMLP and IL-1 (2-fold, P < 0.05 in both cases). These results demonstrate that there is a complement-independent impairment of migration of neutrophils from patients with PNH which may be related to their failure to express GPI-linked proteins involved in cell migration and/or adhesion such as the uPA receptor and the CD66b antigen.

Granulocytes isolated from whole blood of 10 volunteers with Hypaque/Ficoll or Percoll gradient centrifugation showed a significant inhibition of their chemotactic response when incubated with diatrizoate. The influence of 4 different i.v. injected radiographic contrast media (CM) on granulocyte chemotaxis was evaluated using the under agarose assay. Each CM was injected in 10 consecutive outpatients referred for urography. Cells and serum were obtained before and 20 min after the injection of CM. Autologous serum was used as chemoattractive agent, and the cell suspensions from both samples were tested against serum drawn both before and after the injection of CM. No significant changes in granulocyte chemotaxis or the chemotactic potential of serum were seen. Furthermore, there was no change in the chemotactic response towards homologous serum or N-fmlp after injection of diatrizoate or iohexol. In conclusion, the investigation did not unveil any change in granulocyte chemotaxis 20 min after the injection of CM in the 40 subjects studied.

One of the important functions of granulocytes is the ability to respond to a chemoattractive signal by migration. The influence of radiographic contrast media (CM) on the chemoattractive properties of serum was investigated by under agarose technique for chemotaxis. No chemotactic response was seen when serum was incubated with different concentrations of CM after heat inactivation. The CM did not generate the heat stable complement split product C5a-desarg which would have resulted in a chemotactic response. Without heat inactivation all complement available in the serum was activated by the agarose in the chemotaxis assay. Low concentrations of iohexol and iodixanol brought about an increased chemotactic response relative to a reference with saline instead of CM. This may be due to activation of heat labile chemoattractants from other sources than the complement cascade. At high concentrations of all five investigated CM, a decreased number of granulocytes migrated over a shorter distance when compared with the reference, and this may be due to interactions between the CM and chemoattractants or their precursors.

The under agarose method for evaluation of leucocyte chemotaxis was used to investigate the effect of radiographic contrast media (CM) on granulocyte locomotion. The CM tested had no chemoattractive properties. CM inhibited N-fmlp, a synthetic formylated Met-tripeptide, which is a strong chemotactic agent and an analogue to chemotatic peptides produced by bacteria. The inhibition of N-fmlp was most pronounced for diatrizoate. Equiosmolal saline was not so inhibitive. Therefore, some part of the inhibition was caused by factor(s) other than hyperosmolality inherent in the CM solution.