Systemic inflammation and innate immune response in patients with previous anterior uveitis

Abstract

Aim: To determine the presence of systemic inflammation and innate immune responsiveness of patients with a history of acute anterior uveitis but no signs of ocular inflammation at the time of recruitment.

Methods: Tumour necrosis factor alpha (TNF-alpha) production in response to bacterial lipopolysaccharide (LPS) was studied using whole blood culture assay; levels of TNF-alpha in culture supernatants, and soluble interleukin 2 receptor (sIL-2R) in serum were determined by chemiluminescent immunoassay (Immulite); monocyte surface expression of CD11b, CD14, and CD16 and the proportion of monocyte subsets CD14(bright)CD16(-) and CD14(dim)CD16(+) were studied with three colour whole blood flow cytometry; and serum C reactive protein (CRP) levels were determined using immunonephelometric high sensitivity CRP assay.

Results: The CRP level (median, interquartile range) was significantly higher in 56 patients with previous uveitis than in 37 controls (1.59 (0.63 to 3.47) microg/ml v 0.81 (0.32 to 2.09) microg/ml; p=0.008). The TNF-alpha concentration of the culture media per 10(5) monocytes was significantly higher in the patient group than in the control group in the presence of LPS 10 ng/ml (1473 (1193 to 2024) pg/ml v 1320 (935 to 1555) pg/ml; p=0.012) and LPS 1000 ng/ml (3280 (2709 to 4418) pg/ml v 2910 (2313 to 3358) pg/ml; p=0.011). The background TNF-alpha release into the culture media was low in both groups. CD14 expression of CD14(bright)CD16(-) monocytes, defined as antibody binding capacity (ABC), was similar for the patients and controls (22,839 (21,038 to 26,020) ABC v 21,657 (19,854 to 25,646) ABC).

Conclusions: Patients with previous acute anterior uveitis show high innate immune responsiveness that may play a part in the development of ocular inflammation.

Figure 1

Analysis of patients' CD14 and CD16 cell surface markers on CD11b positive monocytes. Aliquots of heparinised blood were triple labelled with fluorescent mAbs, as described in the Methods section. CD11b positive cells were delineated by R1 (A) and monocytes by R2 (B) and a CD14(FL2)/CD16(FL3) dot plot was developed from the monocytes (that is, the events co-locating in R1 and R2) (C). The proportion of CD14^dimCD16⁺ monocytes was 13.4%. CD14 expression, median of the number of PE molecules bound by cells; CD16 expression, relative fluorescence units (RFU); upper horizontal zone, CD14^bright monocytes; middle horizontal zone, CD14^dim monocytes; bottom zone, non-specific PE fluorescence of cells stained with PE conjugated irrelevant mAb; vertical zone on right, CD16⁺ monocytes; vertical zone on left, non-specific PC5 fluorescence.

Figure 2

Relation between the monocyte count and the TNF-α level (A) and the TNF-α level standardised by the monocyte count (B). A 100 μl aliquot of whole blood diluted with RPMI 1640 was cultured with Escherichia coli lipopolysaccharide (10 ng/ml), and the TNF-α level in the culture supernatant was measured as described in the Methods section. Closed circles, patients (n=56); open circles, healthy volunteers (n=37); lines were estimated by a locally weighted scatter plot smoother (LOWESS). Three patients were on antirheumatic drugs and indicated in the figure as follows: ▴, aurothiomalate; ▪, hydroxychloroquine; ▾, sulphasalazine.

Figure 3

TNF-α levels in whole blood culture supernatants. The difference between the patients' cell supernatants (n=56) and control cell supernatants (n=37) was statistically significant (Mann-Whitney U test) at 1000 ng/ml (p=0.011) and 10 ng/ml (p=0.012). LPS, Escherichia coli lipopolysaccharide. Open circles, outliers.