Recent reports have suggested critical roles of myeloid cells in tumor invasion and metastasis, although these findings have not led to therapeutics. Using a mouse model for liver dissemination, we show that mouse and human colon cancer cells secrete CCchemokine ligands CCL9 and CCL15, respectively, and recruit CD34 + Gr-1 − immature myeloid cells (iMCs). They express CCL9/15 receptor CCR1 and produce matrix metalloproteinases MMP2 and MMP9. Lack of the Ccr1, Mmp2, or Mmp9 gene in the host dramatically suppresses outgrowths of disseminated tumors in the liver. Importantly, CCR1 antagonist BL5923 blocks the iMC accumulation and metastatic colonization and significantly prolongs the survival of tumor-bearing mice. These results suggest that CCR1 antagonists can provide antimetastatic therapies for patients with disseminated colon cancer in the liver.C olon cancer is one of the leading causes of cancer-related deaths (1). Although most primary tumors can be resected surgically, colorectal cancer frequently spreads to the liver, which is responsible for the high mortality of the disease (2). For successful metastasis, cancer cells need to invade surrounding tissues, penetrate microvessels, survive in circulation, disseminate to distant organs, form micrometastases, and expand into macrometastases. To progress through these steps, tumor cells often acquire the capability of survival and invasion by activating metastatic signaling pathways or inactivating metastasis suppressor genes (2, 3). In addition to these cell autonomous changes, tumor stromal cells, especially bone marrow-derived myeloid cells, actively participate in early steps of the metastatic cascade in some mouse models (4). For example, tumor-associated macrophages (TAMs) promote migration and intravasation of mammary tumor cells (5, 6). Bone marrow-derived cells that express myeloid cell marker CD11b and granulocyte marker Gr-1 (CD11b + Gr-1 + ) also promote metastasis of breast cancer cells, likely through promotion of intravasation and suppression of immune responses (7). Furthermore, CD11b + myeloid cells that express vascular endothelial growth factor receptor 1 (VEGFR1) accumulate at the metastatic sites before the arrival of lung cancer and melanoma cells and foster the dissemination of the cancer cells (8). These reports suggest that bone marrow-derived myeloid cells can help cancer epithelium in early steps of metastasis. It remains to be determined whether therapeutics targeting such myeloid cells can prevent cancer metastasis (9).As a model for invasive colon cancer, we previously constructed cis-Apc +/Δ716 Smad4 +/− (Apc/Smad4) mice that develop intestinal adenocarcinomas with marked invasions by loss of Apc and Smad4 tumor suppressor genes in the intestinal epithelium (10, 11). In the Apc/Smad4 tumors, we reported that the invading cancer epithelium is associated with immature myeloid cells (iMCs) that express myeloid progenitor cell marker CD34 and CD11b (12). Because these iMCs do not express Gr-1 or VEGFR1, they belong to a different subc...

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The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

Diabetic nephropathy is associated with interstitial macrophage infiltrates, but their contribution to disease progression is unclear. We addressed this question by blockade of chemokine receptor (CCR)1 because CCR1 mediates the macrophage recruitment to the renal interstitium. In fact, when CCR1 was blocked with BL5923, a novel orally available CCR1 antagonist, the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with advanced diabetic nephropathy. Likewise, BL5923 (60 mg/kg, twice a day) orally administered from months 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, transforming growth factor-beta1, and collagen I-alpha1 when compared with untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for interstitial macrophages for tubulointerstitial injury, loss of peritubular microvasculature, interstitial inflammation, and fibrosis in type 2 diabetic db/db mice. These data identify oral treatment with the CCR1 antagonist BL5923 as a potential therapy for late-stage diabetic nephropathy.

The interleukin-1 beta-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 beta precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.