Both Th1 and Th17 are immunopathogenic but differ in other key biological activities

Abstract

The role of Th17 lymphocytes in immunopathogenic processes has been well established, but little is known about their basic cell features. In this study, we compared polarized Th1 and Th17 for key biological activities related to pathogenicity and trafficking. Th1 and Th17 lineages were derived from TCR-transgenic CD4 murine cells specific against hen egg lysozyme. When adoptively transferred into mice expressing hen egg lysozyme in their eyes, both Th1 and Th17 induced ocular inflammation but with slight differences in histological pathology. PCR analysis revealed selective expression of IFN-gamma or IL-17 in eyes of Th1 or Th17 recipients, respectively. Additionally, Th1 and Th17 were found to differ in three other key activities: 1) Th17 cells were inferior to Th1 cells in their capacity to trigger massive lymphoid expansion and splenomegaly; 2) the proportion of Th1 cells among infiltrating cells in inflamed recipient eyes declined rapidly, becoming a minority by day 7, whereas Th17 cells remained in the majority throughout this period; and 3) remarkable differences were noted between Th1 and Th17 cells in their expression of certain surface markers. In particular, reactivated Th1 expressed higher levels of CD49d and alpha(4)beta(7) (mucosal homing) in vitro and higher levels of CXCR3 (Th1 trafficking) in vivo. Reactivated Th17, however, expressed higher levels of alpha(E)beta(7) (epithelial tissue homing) and CD38 (activation, maturation and trafficking) in vitro, but in vivo Th17 expressed higher levels of alpha(4)beta(7) and CCR6 (lymphocyte trafficking). These data reveal that Th1 and Th17 cells differ in several key biological activities influencing migration and pathogenic behavior during inflammatory disease.

FIGURE 1

Polarized and reactivated Th1 and Th17 cells selectively express IFN-γ or IL-17. A, Supernatants of Th1 or Th17 cultures were collected following reactivation and were then tested for levels of IFN-γ or IL-17 by SearchLight technology. The data are expressed as mean values ± SEM of three experiments. B, Intracellular expression of IFN-γ and IL-17 by polarized and reactivated Th1 and Th17 before adoptive transfer into recipient mice. C, Selective expression of IFN-γ or IL-17 transcripts from polarized and reactivated Th1 or Th17, respectively. Total RNA was collected from reactivated cells, pooled, and then analyzed by real time RT-PCR. The data are expressed as mean values ± SEM of two experiments.

FIGURE 2

Polarized Th1 and Th17 cells both induce pathological changes in eyes of recipient mice. A, Immunopathogenicity of polarized Th1 and Th17 cells. Different numbers, as indicated, of polarized and reactivated Th1 or Th17 cells were injected into naive HEL-Tg recipients. Eyes were collected on day 7 and histopathological changes were scored on a scale of 0–9, as detailed in Ref . B–E, Eye sections of a control naive HEL-Tg mouse eye and typical changes in eyes of recipients injected with 1 × 10⁶ polarized and reactivated Th1 or Th17 cells. B, Whole eyes showing inflammatory changes in both the anterior and posterior segments of recipient eyes consisting of mixed inflammatory cellular infiltrates and proteinaceous exudates. C, Corneas of the same two recipient eyes showing edema of the corneal epithelium (e) and stroma (s) in the Th17 recipient. D, Higher magnification depicting infiltration of inflammatory cells (arrow) into the central portion of the cornea in the Th17 recipient. E, Posterior segments demonstrating inflammatory cells in the vitreous (arrowheads) and inner retinal layers, including the nerve fiber layer and ganglion cell layer (arrows). In addition, the Th1 recipient eye shows retinal folding (H&E staining). Original magnification: ×25 (B); ×200 (C); ×630 (D); ×100 (E).

FIGURE 3

IFN-γ and IL-17 transcripts are selectively expressed in inflamed eyes of recipient mice. Eyes of recipient mice were collected on day 5 postadoptive transfer of 2 × 10⁶ reactivated Th1 or Th17 cells. Total RNA was collected, pooled, and analyzed by real time RT-PCR for transcript levels of IFN-γ and IL-17. The data are expressed as mean values ± SEM of two experiments.

FIGURE 4

Th1 and Th17 cells differ in their capacity to stimulate lymphoid expansion in recipient mice. Groups of two or three syngeneic WT mice were injected with 10 × 10⁶ polarized and reactivated Th1 or Th17 cells. Recipient mice were euthanized 4 days later, representative spleens were chosen for photography (A), and spleens were pooled for weight (B) and cell counts (C). In addition, blood samples were collected and pooled and the number of MNLs was determined (D). All values are expressed as the fold increase in recipient mice compared with noninjected controls; injection of naive CD4 cells from 3A9 donors had no effect on the parameters shown here (Ref. and data not shown). The data are expressed as mean values ± SEM of four or five experiments.*, p < 0.05;**, p < 0.0005;***, p < 0.0001.

FIGURE 5

Differential surface marker expression on Th1 and Th17 cells in vitro. A, Suspensions of naive CD4 cells, as well as Th1 or Th17 cells at two different stages in vitro, were immunostained with mAbs against major surface markers. Th1 and Th17 cell surface markers were analyzed at the following culture stages: 1) after polarization and reactivation, just before injection into recipient mice (“React”); and 2) after 11 days of additional culture in the presence of IL-2 (“Expand II”). Numbers represent the percentage of positive or “high” positive cells gated on the CD4⁺1G12⁺7AAD⁻ population. Recorded values were obtained in a representative experiment; similar results were obtained in two additional experiments. B, Flow cytometric plots of a representative experiment showing Th1 and Th17 surface marker expression at the “reactivated” stage.

FIGURE 6

Differential surface marker expression on donor cells in the spleens and eyes of recipient mice. Cells were isolated on day 4 postadoptive transfer of 10 × 10⁶ reactivated Th1 or Th17 cells into recipient mice. Numbers represent the percentage of positive or “high” positive cells gated on the CD4⁺1G12⁺7AAD⁻ population. Recorded values were obtained in a representative experiment; similar data were obtained in an additional experiment.

FIGURE 7

Th1 and Th17 differ in their kinetics of accumulation in recipient mouse eyes and the recruitment of host cells. Recipient mice were injected with 10 × 10⁶ reactivated Th1 or Th17 cells and eyes were collected on days 4 and 7 postadoptive transfer. Cells were obtained by collagenase treatment of the collected eyes, and flow cytometric analysis was performed to measure the percentages of CD4 and 1G12 expression. 1G12 is a clonotypic Ab that selectively stains donor cells. A, Flow cytometry plots from a representative experiment. B, Summary of mean percentage values ± SEM of three experiments.