Key Points Alternatively polarized macrophages are abundant constituents of the tumor microenvironment in T-cell lymphoproliferative disorders. GATA-3 expression identifies a subset of PTCL, NOS with a distinct cytokine profile and inferior survival.

Objective Aminopeptidase N (CD13, EC 3.4.11.2) is a metalloproteinase expressed by fibroblast like synoviocytes (FLS). It has been suggested that CD13 can act chemotactically for T cells in rheumatoid arthritis (RA). The goals of this study were to measure CD13 in vivo and in vitro-in RA samples, and to determine whether CD13 could play a role in homing of T cells to the RA joint. Methods IL-17 treated FLS were used to immunize mice, from which a novel anti-human CD13 monoclonal antibody (591.1D7.34) was developed. 1D7 and a second anti-CD13 monoclonal, WM15, were used to develop a novel ELISA for CD13, and CD13 enzymatic activity was measured in parallel. Chemotaxis of cytokine activated T cells (Tck) was measured by an under-agarose assay. Result We detected substantial amounts of CD13 in synovial fluids, sera, FLS lysates, and culture supernatants by ELISA, with a significant increase in CD13 in RA synovial fluids when compared to osteoarthritis (OA). CD13 accounted for most but not all of the CD13-like enzymatic activity in synovial fluid. Recombinant human CD13 was chemotactic for Tck through a G-protein-coupled-receptor and contributed to the chemotactic properties of synovial fluid independently of enzymatic activity. Conclusion CD13 is released from FLS into culture supernatants and is found in synovial fluid. CD13 induces chemotaxis of Tck, a T cell population similar to that found in RA synovium. This data suggest that CD13 could play an important role as a T cell chemoattractant, in a positive feedback loop that contributes to RA synovitis.

The calcitonin/calcitonin gene-related peptide (CT/ CGRP) gene is selectively transcribed in thyroid C cells and neurons. We have previously shown that the rat CT/CGRP cell-specific enhancer is synergistically regulated by a helix-loop-helix (HLH) protein and the OB2 octamer-binding protein. In this report, we show that the HLH-OB2 enhancer is required for full promoter activity, even in the context of other HLH elements.Since this enhancer appears to be a major controlling element, we have characterized the HLH and OB2 DNA binding proteins. We have identified the major HLH complex as a heterodimer of the ubiquitous upstream stimulatory factor (USF)-1 and USF-2 proteins. USF bound the enhancer with a reasonably high affinity (K D 1.6 nM), comparable to other genes. Characterization of a series of mutations revealed that a portion of the HLH motif is also recognized by OB2 and confirmed that HLH activity requires OB2. We have shown that OB2 is a single DNA binding protein based on UV cross-linking studies. The 68-kDa protein-DNA complex was detected only in C cell lines, including a human C cell line that has robust HLH-OB2 enhancer activity. These results suggest that the calcitonin/CGRP gene is controlled by the combinatorial activity of a ubiquitous USF HLH heterodimer and an associated cell-specific activator.

Connector enhancer of KSR (CNK) is a multidomain protein that participates in Ras signaling in Drosophila eye development. In this report we identify the human homologue of CNK, termed CNK2A, and a truncated alternatively spliced variant, CNK2B. We characterize CNK2 phosphorylation, membrane localization, and interaction with Ras effector molecules. Our results show that MAPK signaling appears to play a role in the phosphorylation of CNK2 in vivo. CNK2 is found in both membrane and cytoplasmic fractions of the cell. In MDCK cells, full-length CNK2 is localized to the lateral plasma membrane. Consistent with previous reports, we show CNK2 interacts with Raf. CNK2 interaction was mapped to the regulatory and kinase domains of Raf, as well as to the carboxyl-terminal half of CNK2. CNK2 also interacts with the Ral signaling components, Ral GTPase, and the RalGDS family member Rlf. CNK2 interaction was mapped to the GEF domain of Rlf. The ability of CNK2 to interact with both Ras effector proteins Raf and Rlf suggests that CNK2 may integrate signals between MAPK and Ral pathways through a complex interplay of components.

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