Decreased polymorphonuclear leukocyte deformability in NIDDM.

OBJECTIVE: To determine the rheological properties of polymorphonuclear leukocytes (PMN) from non-insulin-dependent diabetes mellitus (NIDDM) patients. RESEARCH DESIGN AND METHODS: The deformability of PMN from 33 NIDDM subjects, 13 with impaired glucose tolerance (IGT), and 22 with normal glucose tolerance (NGT) was studied. A Cell Transit Analyzer that measures the transit time of PMN through 8-microns pores was used. Studies were performed under three different conditions: 1) basal state; 2) after incubation with cytochalasin B (20 microM) to dissociate f-actin from the cytoskeleton; and 3) following activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 nM). RESULTS: PMN from diabetic patients were more rigid (i.e., had longer transit time) than those from subjects with NGT or IGT under basal conditions and after cytochalasin B, but not after stimulation with fMLP. The deformability of PMN from subjects with IGT was similar to those of the NGT group. In the pooled data, basal transit time correlated with age; systolic and diastolic blood pressure; HbA1c; and serum creatinine, cholesterol, and triglyceride concentrations (r = 0.29, 0.34, 0.37, 0.48, 0.25, 0.36, 0.29, respectively, P < 0.05 for each). Hypertensive diabetic patients had less deformable PMN than normotensive ones. No relation was found between PMN deformability and the duration of diabetes, type of treatment, or the presence of retinopathy. CONCLUSIONS: These data indicate increased rigidity of PMN in NIDDM that may contribute to development of microcirculatory disturbances and microangiopathy.

Kinetics of granulocyte deformability following exposure to chemotactic stimuli.

In order to quantitate the kinetics of granulocyte deformability changes consequent to chemotactic stimulation, transit times (TT) of neutrophils through 8-microns diameter pores were studied via a modified cell transit analyzer (CTA). Cells isolated from normal human blood were tested at 20-second intervals for 5 minutes following stimulation with N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) or zymosan-activated plasma (ZAP). The effects of cytochalasin B (CB), N-ethylmaleimide (NEM), and ibuprofen were also evaluated. Salient results included: (1) greater TT with increasing fMLP concentration (0.01-100 nM) with a peak response at 40-60 seconds followed by a return to or toward control at 5 minutes; (2) longer TT at 60 seconds for at least 75% of the cells at all fMLP concentrations, yet for 1 nM at 5 minutes nearly one half had TT less than unstimulated cells; and (3) similar temporal responses during a 5-minute period to ZAP simulation, with a nonlinear relation between cell rigidity and F-actin content. CB (20 microM) and NEM (1 mM) caused an immediate 30% to 35% decrease of TT for unstimulated cells; ibuprofen (10-1000 micrograms/ml) did not affect unstimulated TT, yet significantly reduced the response to fMLP and ZAP. Cell volume, as judged by CTA pulse height, decreased following fMLP stimulation, thus indicating that cell swelling does not contribute to the longer pore transit times of activated granulocytes. Our results, combined with literature reports describing microvascular occlusion by neutrophils, strongly suggest the importance of kinetic rather than static studies of granulocyte deformability; further, they indicate the usefulness of the modified CTA for such measurements.

Platelet-induced granulocyte aggregation in vitro.

Recent studies have shown that the response of granulocytes to stimulation is increased by the presence of platelets. Using an assay based on counting aggregates with a Coulter Counter, we found that heparinized or citrated platelet-rich plasma directly caused aggregation of isolated granulocytes. Platelets themselves were frequently present within the granulocyte aggregates. The degree of aggregation depended on the platelet:granulocyte ratio, and increased over a 2--5 min period of mixing, with a slower increase thereafter. Aggregation was calcium-dependent, increased by stimulation of the platelets with ADP or collagen but not serotonin, and only slightly reduced by a thrombin inhibitor (hirudin, at concentrations up to 0.9 U/ml). A similar adhesive interaction in vivo might be relevant to thrombotic and ischaemic pathology.