Yura RE, Bradley SG, Ramesh G, Reeves WB, Bond JS. Meprin A metalloproteases enhance renal damage and bladder inflammation after LPS challenge. Am J Physiol Renal Physiol 296: F135-F144, 2009. First published October 29, 2008 doi:10.1152/ajprenal.90524.2008.-Meprin metalloproteases, composed of ␣ and/or ␤ subunits, consist of membrane-bound and secreted forms that are abundantly expressed in proximal tubules of the kidney as well as secreted into the urinary tract. Previous studies indicated that meprin metalloproteases play a role in pathological conditions such as ischemic acute renal failure and urinary tract infection. The aim of this work was to examine the role of meprins in endotoxemic acute renal failure using meprin ␣ knockout (␣KO), meprin ␤ knockout (␤KO), and wild-type (WT) mice. Differences among the responses of the genotypes were observed as early as 1 h after challenge with 2.5 mg/kg ip Escherichia coli LPS, establishing roles for meprins in the endotoxemic response. Meprin ␣KO mice displayed lower blood urea nitrogen levels and decreased nitric oxide levels, indicative of a decreased systemic response to LPS compared with WT and meprin ␤KO mice. Serum cytokine profiles showed lower levels of IL-1␤ and TNF-␣ in the meprin ␣KO mice within 3 h after LPS challenge and confirmed a role for meprins in the early phases of the host response. Meprin ␣KO mice were also hyporesponsive to LPS administered to the bladder, exhibiting significantly less bladder edema, leukocyte infiltration, and bladder permeability than WT mice. These data indicate that meprin A contributes to the renal and urogenital pathogenesis of endotoxicity. knockout mice; lipopolysaccharide; kidney; metalloproteinase ACUTE RENAL FAILURE (ARF) is a frequent complication of sepsis, a systemic response to infection that often leads to shock and multiple organ failure (49). Intraperitoneal (ip) administration of endotoxin or LPS, a glycolipid component of the gram negative bacterial cell wall, generates a robust host response similar to that observed during sepsis, including elevated blood urea nitrogen (BUN) and creatinine levels, extensive production of proinflammatory mediators (e.g., TNF-␣, IL-1␤), and release of the vasodilator nitric oxide (NO) (15,20,29,54). TNF-␣ is an important mediator of LPSinduced ARF and is also implicated in induction of hypothermia, a hallmark of the rodent response to endotoxemia and a reliable parameter for evaluating the acute effects of LPS (10,19,20,34,37). The injurious effects of LPS are also readily apparent on transurethral administration of LPS, which produces a localized inflammatory response that resembles urinary tract infection (UTI) and consists of extensive leukocyte infiltration, edema, and urothelial disruption in the bladder tissue (17, 43).Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules as well as secreted into the urinary tract (12, 45). They are also expressed in the intestine, skin, and discrete populations of leukocytes (5,12,...

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly(100) and Ser(101) on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.

B-cell maturation antigen (BCMA), a member of the TNF receptor superfamily, serves as the cell surface receptor for B-cell activating factor (BAFF). Upon binding of BAFF to BCMA, an intracellular signaling cascade is initiated resulting in upregulation of JNK pathway signaling events and NFkB mediated transcription. The canonical expression pattern of BCMA begins on germinal center B-cells and becomes maximally expressed on mature cells such as plasma cells. Given the high degree of expression of BCMA on multiple myeloma (MM) cells, the malignant counterpart to plasma cells, it has become a target of interest for CAR T and antibody mediated modalities such as antibody drug conjugates and bispecific molecules. Recent clinical data from clinical trials employing BCMA targeted CAR T cells or T cell engager (TCE) antibodies have demonstrated significant responses in heavily pretreated myeloma patients with overall response rates ranging from 70% to 80%. As BCMA is known to be expressed in earlier B-cell lineages, we sought to explore the expression of BCMA in non-Hodgkin lymphoma (NHL) and its sensitivity to CC-93269, a 2+1 TCE currently being clinically investigated in MM. NHL is a heterogeneous collection of lymphomas that can be classified into two major subgroups; aggressive lymphomas of which diffuse large B-cell (DLBCL) is the most prevalent subtype and indolent lymphomas of which follicular lymphoma is the largest constituent. We first sought to directly quantitate cell surface expression of BCMA utilizing a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In a panel of 43 NHL cell lines, we determined that BCMA expression ranged from 43 to 17,048 molecules per cell (median, 420). An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) (15,866 molecules/cell) served as negative and positive controls, respectively. Additionally, the MM cell line H929 was profiled to serve as an additional control with a BCMA expression level of 7,065 molecules/cell. Next, utilizing quantitative PCR we found that relative BCMA mRNA expression in the lymphoma cell lines ranged from 0.001 to 0.17-fold when normalized to the H929 MM cell line. Furthermore, we were able to determine that in the lymphoma cells there is a poor correlation between protein expression (mean fluorescent intensity) and mRNA expression (r2, 0.33). We next examined if there was any correlation between BCMA surface expression and T-cell mediated cytotoxicity after administration of CC-93269 in a co-culture assay. We selected 11 DLBCL cell lines with a surface expression ranging from 45 molecules to 17,000 molecules per cells and incubated them in a co-culture system with a defined 1:5 target:effector ratio with CC-93269 (0-200 ng/ml) for five days. Significant apoptosis as measured by annexin V and ToPro-3 staining of CFSE positive target cells was observed in 10 of the 11 cell lines profiled with an IC50 of 0.1 to 38 ng/ml for CC-93269. As controls, the K562 isogenic pair were also profiled with the K562-BCMA cell line exhibiting an IC50 of 0.5 ng/ml and no activity observed against the parental K562 cell line. Additionally, a bispecific antibody where the two binding domains for BCMA was altered to target HEL (hen egg lysozyme) demonstrated no activity against any of the cell lines profiled at a defined dose of 200 ng/ml. No association between CC-93269 activity and BCMA expression or cell of origin was found. To determine the expression of BCMA in primary DLBCL biopsy samples, we developed a novel monoclonal BCMA immunohistochemistry antibody (clone: G12). The antibody and IHC staining protocol were validated to have good on-target specificity in both cell lines and tissues, including MM and DLBCL biopsies, with a range of stain intensity (1-3+) observed in both the golgi and on the plasma membrane. A proof of concept study on a cohort of 110 commercial DLBCL samples is currently underway. Cumulatively, our data demonstrate that BCMA is expressed on the cell surface of a broad panel of NHL cell lines and in primary DLBCL lymph node biopsies. Additionally, the expression levels of BCMA in these preclinical cell line models was sufficient to elicit significant CC-93269 mediated cytotoxicity. These data highlight the potential for the treatment of DLBCL patients with a 2+1 T-cell engager targeting BCMA. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties. Gray:Celgene: Employment, Equity Ownership. Yura:Celgene: Employment, Equity Ownership. Hersey:Celgene: Employment, Equity Ownership. Chan:Celgene: Employment, Equity Ownership. Zhang:Celgene: Employment, Equity Ownership. Boss:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

Meprins, zinc metalloproteases that are expressed in kidney proximal tubules, intestinal epithelium, and leukocytes, have been implicated in the host response to infection. For example, we have observed high levels of urinary meprin associated with active urinary tract infections (UTIs) in humans. To determine whether meprins affect the course of UTIs, meprin α knockout (KO) and wild‐type (WT) mice were infected with uropathic Escherichia coli via catheterization. Initial studies indicate that bacterial counts in the bladders of meprin α KO mice are approximately ten times greater than in WT mice after 48h, indicating a role for meprin in response to infection. To further explore the role of meprins in the immune response, meprin α KO and WT mice were challenged with IP injection of lipopolysaccharide (LPS). After 24h, blood urea nitrogen levels were markedly lower and there was less hypothermia in the meprin α KO compared to WT mice, indicating that meprins are involved in modulation of the immune response. Additional experiments demonstrated that soluble meprin A disrupts tight junctions of cultured MDCK cells as revealed by immunocytochemical staining for occludin and ZO‐1. Taken together, the results indicate that meprins enable leukocytes to gain more ready access to the urinary tract, thereby promoting resolution of the infection. (NIH Grant DK 19691)