Cytokines are critical for normal cell growth and immunoregulation but also contribute to growth of malignant cells and drive immune-mediated disease. A major subset of immunoregulatory cytokines, roughly 60, use the type I and type II cytokine receptors and pharmacological targeting of these cytokines/cytokines receptors has proven to be efficacious in treating immune and inflammatory diseases. These receptors rely on Janus family of kinases (Jaks) for signal transduction and recently the first Jak inhibitor has been approved by the FDA. Many other Jakinibs are likely to follow and in this brief review, we will discuss the state-of-the art of this new class of pharmacological agents.

There are multiple drivers of leukocyte recruitment in lung allografts that contribute to lymphocytic bronchitis (LB) and bronchiolitis obliterans (BO). The innate mechanisms driving (or inhibiting) leukocyte trafficking to allografts remain incompletely understood. This study tested the hypothesis that CD73 (ecto-5′ nucleotidase), an enzyme that catalyzes the conversion of AMP to adenosine, is a critical negative regulator of LB and BO. Implantation of tracheal allografts from wild type (WT) mice into CD73−/− recipients revealed a striking increase in airway luminal obliteration at 7 d (62 ± 4% and 47 ± 5% for CD73−/− and WT allograft recipients, respectively; p = 0.046). There was also a concordant increase in CD3+ lymphocytic infiltration (523 ± 41 cells and 313 ± 43 cells for CD73−/− and WT allograft recipients, respectively; p = 0.013). Because real-time PCR revealed a 43-fold upregulation of mRNA for the adenosine A2A receptor (A2AR) in WT allografts compared with WT isografts (p = 0.032), additional experiments were performed to determine whether the protective effect of CD73 was due to generation of adenosine and its stimulation of the A2AR. Treatment of WT recipients with an A2AR agonist significantly reduced CD3+ lymphocyte infiltration and airway luminal obliteration; similar treatment of CD73−/− recipients rescued them from LB and airway obliteration. These data implicate CD73 acting through adenosine generation and its stimulation of the A2AR as a critical negative modulator of lymphocyte recruitment into airway allografts. The CD73/adenosine axis might be a new therapeutic target to prevent BO.

Introduction

CP-690550 is a small molecule inhibitor of Janus kinase 3 (JAK3), a critical enzyme in the signaling pathway of multiple cytokines (interleukin (IL)-2, -7, -15 and -21) that are important in various T cell functions including development, activation and homeostasis. The purpose of this study was to evaluate CP-690550 in murine collagen-induced (CIA) and rat adjuvant-induced (AA) models of rheumatoid arthritis (RA).

Methods

CIA and AA were induced using standard protocols and animals received the JAK3 inhibitor via osmotic mini-pump infusion at doses ranging from 1.5–15 mg/kg/day following disease induction. Arthritis was assessed by clinical scores in the CIA models and paw swelling monitored using a plethysmometer in the AA model until study conclusion, at which time animals were killed and evaluated histologically.

Results

CP-690550 dose-dependently decreased endpoints of disease in both RA models with greater than 90% reduction observed at the highest administered dose. An approximate ED50 of approximately 1.5 mg/kg/day was determined for the compound based upon disease endpoints in both RA models examined and corresponds to CP-690550 serum levels of 5.8 ng/ml in mice (day 28) and 24 ng/ml in rats (day 24). The compound also reduced inflammatory cell influx and joint damage as measured histologically. Animals receiving a CP-690550 dose of 15 mg/k/d showed no histological evidence of disease.

Conclusion

The efficacy observed with CP-690550 in CIA and AA suggests JAK3 inhibition may represent a novel therapeutic target for the treatment of RA.

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by γc-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.