Christopher A. Gabel

P2 purinergic receptor modulation of cytokine production

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y6 receptor mediated impact on interleukin (IL)-8 production, (2) P2Y11 receptor-mediated affects on IL-12/23 output, and (3) P2X7 receptor mediated IL-1β posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.

Externalization of the Leaderless Cytokine IL-1F6 Occurs in Response to Lipopolysaccharide/ATP Activation of Transduced Bone Marrow Macrophages

An interesting trait shared by many members of the IL-1 cytokine family is the absence of a signal sequence that can direct the newly synthesized polypeptides to the endoplasmic reticulum. As a result, these cytokines accumulate intracellularly. Recent studies investigating IL-1β export established that its release is facilitated via activation of an intracellular multiprotein complex termed the inflammasome. The purpose of the current study was to explore the mechanism by which murine IL-1F6 is released from bone marrow-derived macrophages (BMDMs) and to compare this mechanism to that used by IL-1β. BMDMs were engineered to overexpress IL-1F6 by retroviral transduction; cells overexpressing GFP also were generated to provide a noncytokine comparator. The transduced cells constitutively expressed IL-1F6 and GFP, but they did not constitutively release these polypeptides to the medium. Enhanced release of IL-1F6 was achieved by treating with LPS followed by ATP-induced activation of the P2X7 receptor; GFP also was released under these conditions. No obvious proteolytic cleavage of IL-1F6 was noted following P2X7 receptor-induced release. Stimulus-induced release of IL-1F6 and GFP demonstrated comparable susceptibility to pharmacological modulation. Therefore, transduced IL-1F6 is released in parallel with endogenous mature IL-1β from LPS/ATP-treated BMDMs, but this externalization process is not selective for cytokines as a noncytokine (GFP) shows similar behavior. These findings suggest that IL-1F6 can be externalized via a stimulus-coupled mechanism comparable to that used by IL-1β, and they provide additional insight into the complex cellular processes controlling posttranslational processing of the IL-1 cytokine family.

A NOVEL PROTEIN THERAPEUTIC JOINT RETENTION STRATEGY BASED ON COLLAGEN-BINDING AVIMERS

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross‐linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra‐articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein–protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen‐binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen‐binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra‐articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL‐1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL‐1 receptor. In vitro, IL‐1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra‐articular IL‐1‐induced IL‐6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL‐1Ra_M26 and native IL‐1Ra inhibited IL‐6 output when co‐administered with the IL‐1 challenge, only IL‐1Ra_M26 inhibited when administered 1 week prior to IL‐1 challenge. Collagen‐binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics.