Neutrophils and leucocyte adhesion molecules in sickle cell retinopathy

Abstract

Aims: The expression of the adhesion molecules ICAM-1, VCAM-1, and P-selectin, and the distribution and number of polymorphonuclear leucocytes (PMNs) were investigated in sickle cell retinopathy (SCR) and compared to the normal retina.

Methods: Postmortem ocular tissue was obtained from five subjects (16, 21, 28, 40, and 41 years of age) with sickle haemoglobinopathies and from one control subject. Tissue was cryopreserved, and streptavidin peroxidase immunohistochemistry was performed with antibodies against ICAM-1, VCAM-1, and P-selectin. Immunohistochemical reaction product was scored, and PMN numbers were counted in sections stained with non-specific esterase.

Results: Increased ICAM-1, VCAM-1, and P-selectin immunoreactivities were observed in sickle cell subjects compared to the control subject. The highest ICAM and P-selectin immunoreactivity was associated with intraretinal vessels adjacent to the preretinal neovascular formation in subjects with proliferative retinopathy. This was not the case with VCAM-1 immunoreactivity, which was highest in intraretinal vessels adjacent to the sea fan when the sea fan was still "in statu nascendi." Fully formed, "older" sea fans had the highest levels of VCAM-1. The increase in adhesion molecule immunoreactivity was paralleled by an increase in intraretinal PMNs. The number of intraretinal PMNs increased with progression of the disease and the numbers surpassed those in control subjects by threefold. In the sea fan with the greatest VCAM-1 immunoreactivity, there were 20 times more PMNs were observed than in the rest of the retina in the same subject.

Conclusion: These data suggest that adhesion molecule mediated leucocyte adhesion might play an important part in the vaso-occlusive phase of sickle cell retinopathy and in autoinfarction of sea fan formations.

Figure 1

Sections through portions of retina containing sea fans immunolabelled with anti-perlecan antibody, a marker for viable blood vessels. Sea fans varied in morphology from one featuring primarily large caliper dilated vessels (A) (from case 4), one consisting of small diameter capillary-like vessels (B) (from case 4), and one containing both viable and infarcted blood vessels (C) (from case 2). Infarcted, perlecan-negative blood vessels are indicated by the arrowheads in (C). Perlecan immunoreactivity and haematoxylin counterstain; magnification, (A) and (B) 120×, (C) 80×.

Figure 2

Localisation of VCAM-1 , ICAM-1, and P-selectin in the retina from a control subject (case 6) (left) and from a sickle cell subject (case 4) (right). Upregulation of all three molecules was observed in blood vessels of sickle cell retinas. Magnification 85×.

Figure 3

Comparison between immunohistochemical scores in subjects with proliferative sickle cell retinopathy (cases 1, 2, 4) and the control subject (case 6). Two areas in the retina of case 4 are represented (4A and 4B) and scores for sea fan formations and two areas within retina (perfused central retina and border of perfused and non-perfused peripheral retina) are represented for the sickle cell subjects. (A) Mean (SEM) score for VCAM-1 immunoreactivity. (B) Mean immunohistochemical scores for ICAM-1. (C) Mean immunohistochemical scores for P-selectin.

Figure 4

Immunolocalisation of von Willebrand factor (vWf) (top left), VCAM-1 (top right), ICAM-1 (bottom left), and enzyme histochemical localisation of non-specific esterase (NSE) (bottom right) in the large vessel sea fan shown in Figure 1A. VCAM-1 showed marked immunoreactivity. ICAM-1 immunostaining was relatively weak in sea fans compared to that observed in retinal vessels, but was elevated compared to controls. Numerous PMNs (NSE positive cells were located within the lumen of sea fans. Magnification 120×.

Figure 5

Immunohistochemistry scores for perfused and non-perfused retina for all sickle cell subjects and control subject (case 6). Scores for three areas of subjects 4's eye are presented, inferior and two areas with sea fans (4A and 4B), and a superior (5) and inferior block (5 Inf) from case 5 which had non-proliferative retinopathy. Case 1 was excluded from this analysis because retina was not included in the sample. (A) Mean (SEM) score for VCAM-1 immunoreactivity. (B) Mean immunohistochemical scores for ICAM-1. (C) Mean immunohistochemical scores for P-selectin.

Figure 6

Mean number of polymorphonuclear leucocytes (PMNs) per mm² of retina and sea fans. PMNs were elevated in all sickle cell retinas compared to the control subject (p <0.02 for cases 3, 4B, and 5) and further elevated in sea fan formations compared with control subject, case 6.

Figure 7

Summary of immunohistochemistry scores for sickle cell subjects and control subject. The values for sickle cell subjects represent all of the scores in Figure 5. (A) When all scores for VCAM-1 in sickle cell subjects were combined, there was a significant difference between the control subject's scores and the sea fan scores (*p<0.01), but the difference was not significant for the two areas of sickle cell retina. (B) When ICAM-1 scores for all subjects were combined, there was a significant difference between ICAM-1 in perfused and border areas of the sickle cell retinas compared to control subjects retina (*p<0.05) but the difference between sea fans and control subjects retina was not significant (p=0.1). (C) The P-selectin scores for all areas of sickle cell retina and the sea fans was significantly greater than the score in the control subject (p<0.05).