CD4+ and CD8+ cells were detectable in AH samples from normal eyes, hence these results strongly suggest that CD4+ and CD8+ cells occur constitutively in the eye chambers of the normal canine eye. Presumably, they belong to the pool of Trm cells or represent migratory naïve T cells, or both. However, amounts of the analyzed cells were only slightly above the limit of detection of flow cytometry. Therefore, it should be concluded that in terms of their absolute count, CD4+ and CD8+ cells represent only trace populations of cells in the eye chambers of the normal canine eye. This conclusion is relatively concordant with the outcome of our earlier research, in which we were able to demonstrate the presence of these cells in normal bovine and porcine chambers of the eye [2]. In both of these species, the said cells appeared in very low amounts, although their absolute count was distinctly higher than in dogs (which to some extent can be explained by the fact that the ocular globe of pigs and cows is much bigger than in dogs).
With respect to the percentage of CD4+ cells, much similarity to the results obtained in mice [1] and pigs but not in cattle [2] was achieved in the current study, as in the two former species the single positive CD4+ cells constituted only a trace subset among the lymphocyte population of eye chambers. However, as regards the proportion of CD8+ in the lymphocyte population of the eye chambers, dogs are distinctly different from the other species. The value of this parameter in dogs was by 9-, 4- and 12-fold lower than in mice [1], cattle and pigs [2], respectively. These results are in agreement with our previous conclusion that the proportion of CD4+ and CD8+ subsets in the lymphocyte population of eye chambers can differ considerably between animal species.
In this study, all parameters assayed in AH versus their values in PB were compared. Such comparisons might be considered as redundant because considerable diferences in the composition of the lymphocyte population between PB and the effector sites of immune response (and the eye chambers should be counted as a site of this type) are a natural event. However, such comparisons make it more evident that the proportion of CD4+ and CD8+ cells in the lymphocyte population of the eye chambers of normal eyes is very small; the mean values of both parameters equalled mere 3.66 and 3.28%, respectively, which means that they were by 10- and 8-fold lower, respectively, than the analogous percentages in PB. It can be suspected that such low and specific homing of CD4+ and CD8+ cells to the eye chambers is involved in the induction and maintenance of ocular immune privilege. Let us not ignore the finding arising from our research, i.e. that the combined proportion of CD4+ and CD8+ cells constitutes no more than 7% of the total lymphocyte population of the eye chambers. Determination of what type of other immunocompetent cells hides within this population requires further tests including a wider antibody panel.
Another purpose of confronting the data obtained from AH samples with the corresponding data achieved in PB was to confirm that the small percentage of CD4- and CD8-expressing cells in AH samples was not caused by erroneous labeling of cells, or that the presence of lymphocytes in AH samples was not due to their contamination with blood. The typical distribution of CD4- and CD8-expressing cells in PB samples as well as the fact that this distribution was distinctly different from the one determined in AH samples prove that this aim was achieved. Some doubt may arise whether the presence of lymphocytes in eye chambers might not have been a consequence of their postmortem extravasation from the systemic circulation into eye chambers. However, it that had been the case, the percentage of the analyzed cells would have been very close to the one identified in PB. This line of reasoning is supported by the results concerning the distribution of CD4+ and CD8+ cells in AH sample from a dog which choked on a bone. The percentage, shape and location of both cell subsets in AH were almost the same as those in PB. Furthermore, the number of events collected for combined subsets of CD4+ and CD8+ cells in AH sample of this dog was approximately 12-fold higher (data not shown) than in AH samples from normal eyes. It is common knowledge that strangulation may cause capillary rupture in the conjunctiva and sclera. Therefore, these results strongly suggest that choking may result in passive leakage of lymphocytes across damaged blood vessels into the eye chambers. To the best of our knowledge, this is the first report on this subject.
The mean percentage and absolute count of CD4+ cells in AH samples from cataract eyes were almost 2- and 3-fold greater than in normal eyes, respectively. Nevertheless, these differences were not statistically significant. Hence, there is only enough evidence to claim that there was a certain trend towards an increasing number of CD4+ cells in cataract eyes. The situation looks different in the case of CD8+ cells. The percentage and absolute counts of these cells in the eye chambers of the cataract eyes were increased by about 2.7- and 6-fold, respectively, compared to normal ones, and the differences were significant. These results strongly suggests that in terms of the local immune response, the development of uncomplicated cataract in dogs may not be immunologically neutral, but rather it may be associated with the recruitment of CD8+ cells into the eye chambers. This conclusion implicates that in terms of the presence of CD8+ cells in the eye chambers, the cataract eye may not represent faithfully the condition of a healthy eye, as it is suspected. For reasons discussed in the section “A major limitation of the study”, the currently available research capabilities preclude any deeper interpretation of the cataract-related local CD8+ cell recruitment or the trend demonstrated by CD4+ cells towards this direction, as such phenotypes might hide different types of cells (e.g. naïve, memory, effector or regulatory T cells or even non T cells).
The mean percentage and absolute count of CD4+ cells were increased approximately 3- and 15-fold in uveitic eyes, respectively, compared to normal eyes. The values of corresponding parameters for CD8+ cells were about 3- and 18-fold greater in uveitic eyes than in normal ones. These results indicate that uveitis was associated with the infiltration of the eye chambers by CD8+ and CD4+ T cells. To our knowledge, this is the first report on the evaluation of the numbers of these cells in the eyes chambers of dogs with uveitis. However, this is not an original finding, because it has been widely accepted that T cells play a predominant role in uveitis [12, 16, 27, 28]. The relatively selective recruitment of CD8+ and CD4+ T cells into eye chambers was reported in patients with Fuchs’ heterochromic iridocyclitis and idiopathic anterior uveitis [27], respectively. Considering the literature data [28, 29], it can be assumed that CD8+ and CD4+ cells infiltrating the chambers of uveitic eyes constitute largely proinflammatory T cells.
It should be highlighted that the assessment of the abundance of the analyzed cells in the eye chambers of uveitic eyes was not a research problem per se as set in this study. A group of dogs with uveitis was included so as to confront the scale of cataract- and uveitis-induced changes in evaluated parameters, hence it served as a reference. The mean absolute counts of CD8+ and CD4+ cells in the eye chambers of uveitic eyes were approximately 3- and 5-fold higher, respectively, compared to cataract eyes. Thus, the demonstrated increase in the number of CD8+ cells in the eye chambers of cataract eyes was much smaller than the one occurring in uveitic eyes. The study did not show differences in the CD4/CD8 ratio between any of the experimental groups, which means that CD8+ cells increased proportionally to CD4+ cells. This conclusion confirms our suspicion that in the course of canine cataract there is a tendency towards an increasing number of CD4+ cells in the eye chambers. However, it needs to be emphasized strongly that the scale of this trend was minimal when compared to the impressive increase in the CD4+ cell counts in uveitic eyes.