Differential production of macrophage inflammatory protein 1gamma (MIP-1gamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro and in vivo

Abstract

Due to differential expression of chemokine receptors, the Th1 and Th2 subsets of CD4(+) T cells differ in their migratory responses to chemokines. These differences in the migration patterns are likely to play a role in the initiation and regulation of Th1 and Th2 immune responses, inflammatory processes, and T-cell-mediated pathology. In the present study we evaluated the role of activated Th cells as producers of chemokines. Three different sources of murine Th cells were used, i.e., long-term-cultured Th1 and Th2 cell clones, Th1 and Th2 cells differentiated from naïve CD4(+) spleen and lymph node cells in vitro, and Th1 and Th2 subsets polarized in vivo using a murine experimental Leishmania major infection model. Following stimulation with anti-CD3, macrophage inflammatory protein 1gamma (MIP-1gamma) and lymphotactin were produced selectively by Th1 cells but not by Th2 cells. In contrast, only Th2 cells produced MIP-2. The possible biological relevance of these data was substantiated by the finding that in vivo-polarized Th1 cells, but not Th2 cells, produced MIP-1gamma and lymphotactin while in vivo-polarized Th2 cells secreted MIP-2. The above data demonstrate that Th1 and Th2 cells differ in their ability to produce chemokines, suggesting that Th1 and Th2 subsets differentially contribute to recruitment of cells into inflammatory foci.

FIG. 1.

Differential production of lymphotactin, MIP-1γ, and MIP-2 by Th1- and Th2-cell clones.The Th1-cell clones B10BI, LNC-2, AgB4, and COH-2 and the Th2-cell clones D10.G4.1 and L1/1 were stimulated with immobilized anti-CD3 MAb for 20 h. Total-cell RNA was subjected to RPA, and chemokine release was measured by ELISA. (A) RPA autoradiograms showing the mRNA expression of lymphotactin, MIP-1γ, and MIP-2 and of the corresponding housekeeping gene L32. (B) Lymphotactin, MIP-1γ, and MIP-2 content in SN of activated Th-cell clones. The results are the mean and SD of one representative experiment of three performed. n.d., not done.

FIG. 2.

IFN-γ and IL-4 production by Th1- and Th2-cell clones. The Th1 cell clones B10BI, LNC-2, AgB4, and COH-2 and the Th2-cell clones D10.G4.1 and L1/1 were activated with immobilized anti-CD3 MAb for 20 h. SN were assayed for IFN-γ and IL-4 release by ELISA. The results are the mean and SD of one representative experiment of three performed.

FIG. 3.

IFN-γ and IL-4 release by Th1 and Th2 cells differentiated in vitro. Naïve CD4⁺ T cells were purified from spleens and lymph nodes of C57BL/6 mice and differentiated to Th1 or Th2 phenotypes in vitro as described in Materials and Methods. The cells were restimulated with immobilized anti-CD3 MAb for 24 h, and SN were assayed for IFN-γ and IL-4 using ELISA. The results are the mean and SD of two experiments.

FIG. 4.

CD4⁺ Th cells polarized in vitro differentially release lymphotactin, MIP-1γ, and MIP-2. Naïve CD4⁺ T cells differentiated to either Th1 or Th2 cells in vitro were restimulated with anti-CD3 MAb for 24 h as described in Materials and Methods. Total-cell RNA was analyzed for chemokine mRNA expression by RPA. (A) RPA autoradiograms showing the determination of mRNA expression of lymphotactin and MIP-1γ as well as of the corresponding housekeeping gene L32. (B) The SN were tested for lymphotactin, MIP-1γ, and MIP-2 production by ELISA. The results are the mean and SD of two experiments.

FIG. 5.

In vivo-differentiated Th1 cells secrete lymphotactin and MIP-1γ, whereas Th2 cells produce MIP-2. Resistent C57BL/6 and susceptible BALB/c mice were infected with L. major promastigotes in the hind footpads. At 4 weeks after infection, CD4⁺ T cells were purified from draining LN and restimulated with anti-CD3 MAb for 48 h. SN were assayed for IFN-γ and IL-4 (A) and for lymphotactin, MIP-1γ, and MIP-2 (B) using ELISA. The results are the mean and SD of one representative experiment of three performed.