Publications

For a comprehensive list of  Dr. Walcheck's publications, refer to PubMed, a service provided by the National Library of Medicine.


RECENT PUBLICATIONS:
Front Cell Infect Microbiol. 2017 Apr 25;7:138. doi: 10.3389/fcimb.2017.00138. eCollection 2017.
Ectodomain Shedding by ADAM17: Its Role in Neutrophil Recruitment and the Impairment of This Process during Sepsis.
Mishra HK, Ma J, Walcheck B.

Abstract

Neutrophils are specialized at killing bacteria and are recruited from the blood in a rapid and robust manner during infection. A cascade of adhesion events direct their attachment to the vascular endothelium and migration into the underlying tissue. A disintegrin and metalloproteinase 17 (ADAM17) functions in the cell membrane of neutrophils and endothelial cells by cleaving its substrates, typically in a cis manner, at an extracellular site proximal to the cell membrane. This process is referred to as ectodomain shedding and it results in the downregulation of various adhesion molecules and receptors, and the release of immune regulating factors. ADAM17 sheddase activity is induced upon cell activation and rapidly modulates intravascular adhesion events in response to diverse environmental stimuli. During sepsis, an excessive systemic inflammatory response against infection, neutrophil migration becomes severely impaired. This involves ADAM17 as indicated by increased levels of its cleaved substrates in the blood of septic patients, and that ADAM17 inactivation improves neutrophil recruitment and bacterial clearance in animal models of sepsis. Excessive ADAM17 sheddase activity during sepsis thus appears to undermine in a direct and indirect manner the necessary balance between intravascular adhesion and de-adhesion events that regulate neutrophil migration into sites of infection. This review provides an overview of ADAM17 function and regulation and its potential contribution to neutrophil dysfunction during sepsis.



J Biol Chem. 2017 Apr 14;292(15):6339-6351. doi: 10.1074/jbc.M116.746859. Epub 2017 Feb 23.
Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17.
Buchanan PC, Boylan KLM, Walcheck B, Heinze R, Geller MA, Argenta PA, Skubitz APN.


Abstract
We previously showed that the cell adhesion molecule Nectin-4 is overexpressed in ovarian cancer tumors, and its cleaved extracellular domain can be detected in the serum of ovarian cancer patients. The ADAM (adisintegrin and metalloproteinase) proteases are involved in ectodomain cleavage of transmembrane proteins, and ADAM17 is known to cleave Nectin-4 in breast cancer. However, the mechanism of Nectin-4 cleavage in ovarian cancer has not yet been determined. Analysis of ovarian cancer gene microarray data showed that higher expression of Nectin-4, ADAM10, and ADAM17 is associated with significantly decreased progression-free survival. We quantified Nectin-4 shedding from the surface of ovarian cancer cells after stimulation with lysophosphatidic acid. We report that ADAM17 and ADAM10 cleave Nectin-4 and release soluble Nectin-4 (sN4). Small molecule inhibitors and siRNA knockdown of both ADAM proteases confirmed these results. In matched samples from 11 high-grade serous ovarian cancer patients, we detected 2-20-fold more sN4 in ascites fluid than serum. Co-incubation of ovarian cancer cells with ascites fluid significantly increased sN4 shedding, which could be blocked using a dual inhibitor of ADAM10 and ADAM17. Furthermore, we detected RNA for Nectin-4, ADAM10, and ADAM17 in primary ovarian carcinoma tumors, secondary omental metastases, and ascites cells isolated from serous ovarian cancer patients. In a signaling pathway screen, lysophosphatidic acid increased phosphorylation of AKT, EGF receptor, ERK1/2, JNK1/2/3, and c-Jun. Understanding the function of Nectin-4 shedding in ovarian cancer progression is critical to facilitate its development as both a serum biomarker and a therapeutic target for ovarian cancer.


Elife. 2016 Dec 8;5. pii: e17375. doi: 10.7554/eLife.17375.
Tumor-induced MDSC act via remote control to inhibit L-selectin-dependent adaptive immunity in lymph nodes.
Ku AW, Muhitch JB, Powers CA, Diehl M, Kim M, Fisher DT, Sharda AP, Clements VK, O'Loughlin K, Minderman H, Messmer MN, Ma J, Skitzki JJ, Steeber DA, Walcheck B, Ostrand-Rosenberg S, Abrams SI, Evans SS.

Abstract

Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive network that drives cancer escape by disabling T cell adaptive immunity. The prevailing view is that MDSC-mediated immunosuppression is restricted to tissues where MDSC co-mingle with T cells. Here we show that splenic or, unexpectedly, blood-borne MDSC execute far-reaching immune suppression by reducing expression of the L-selectin lymph node (LN) homing receptor on naïve T and B cells. MDSC-induced L-selectin loss occurs through a contact-dependent, post-transcriptional mechanism that is independent of the major L-selectin sheddase, ADAM17, but results in significant elevation of circulating L-selectin in tumor-bearing mice. Even moderate deficits in L-selectin expression disrupt T cell trafficking to distant LN. Furthermore, T cells preconditioned by MDSC have diminished responses to subsequent antigen exposure, which in conjunction with reduced trafficking, severely restricts antigen-driven expansion in widely-dispersed LN. These results establish novel mechanisms for MDSC-mediated immunosuppression that have unanticipated implications for systemic cancer immunity.


PLoS One. 2015 Mar 27;10(3):e0121788. doi: 10.1371/journal.pone.0121788.

Identification of an ADAM17 Cleavage Region in Human CD16 (FcγRIII) and the Engineering of a Non-Cleavable Version of the Receptor in NK Cells.
Jing Y, Ni Z, Wu J, Higgins L, Markowski TW, Kaufman DS, Walcheck B.

Abstract

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively.  Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli.  We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1¢ positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16.  Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays.  We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells.  CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells.  Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.



J Leukoc Biol. 2015 Mar;97(3):447-54. doi: 10.1189/jlb.3HI0714-340R. 
Regulation of CXCR2 expression and function by A Disintegrin And Metalloprotease-17 (ADAM17)
Hemant K Mishra, Chunmei Long, Nooshin S. Bahaie, and Bruce Walcheck

Abstract

The chemokine receptor CXCR2 is expressed at high levels on circulating neutrophils and is critical for directing their migration to sites of inflammation.  CXCR2 surface levels are rapidly modulated by two mechanisms, cell internalization and recycling upon ligand binding and by a metalloprotease activity following overt neutrophil activation by non-ligand stimuli.  The latter process has only been described in human neutrophils, and essentially nothing is known about its functional relevance and the specific protease involved.  We show that targeting ADAM17 in mouse and human neutrophils blocks CXCR2 down-regulation induced by non-ligand stimuli but not by chemokine ligands.  This was determined using a selective ADAM17 inhibitor, an ADAM17 function blocking antibody, and ADAM17 gene-targeted mice.  CXCR2 is known to undergo a marked down-regulation during various inflammatory disorders, and this is associated with impaired neutrophil recruitment.  We show that blocking ADAM17 activity reduced CXCR2 down-regulation on circulating neutrophils and enhanced their recruitment during acute inflammation, which was reversed by a CXCR2 inhibitor.  Taken together, our findings demonstrate that unlike CXCR2 internalization, ADAM17 induction down-regulates the receptor in an irreversible manner and may serve as a master switch in controlling CXCR2 function, but may also contribute to neutrophil dysfunction during excessive inflammation.    



Exp Hematol. 2015 Jan;43(1):44-52.e1-3. doi: 10.1016/j.exphem.2014.10.001.
ADAM17 limits the expression of CSF1R on murine hematopoietic progenitors.
Becker AM, Walcheck B, Bhattacharya D.

Abstract

All-lymphoid progenitors (ALPs) yield few myeloid cells in vivo, but readily generate such cells in vitro. The basis for this difference remains unknown. We hypothesized that ALPs limit responsiveness to in vivo concentrations of myeloid-promoting cytokines by reducing expression of the corresponding receptors, potentially through post-transcriptional mechanisms. Consistent with such a mechanism, ALPs express higher levels of Csf1r transcripts than their upstream precursors, yet show limited cell surface protein expression of CSF1R. ALPs and other hematopoietic progenitors deficient in ADAM17, a metalloprotease that can cleave CSF1R, display elevated cell surface CSF1R expression. Adam17-/- ALPs, however, fail to yield myeloid cells upon transplantation into irradiated recipients. Moreover, Adam17-/- ALPs yield fewer macrophages in vitro than control ALPs at high concentrations of M-CSF. Mice with hematopoietic-specific deletion of Adam17 have grossly normal numbers of myeloid and lymphoid progenitors and mature cells in vivo. These data demonstrate that ADAM17 limits CSF1R protein expression on hematopoietic progenitors, but that compensatory mechanisms prevent elevated CSF1R levels from altering lymphoid progenitor potential.



Prostate. 2014 Jul;74(10):1059-67. doi: 10.1002/pros.22826. 
Increased expression of GCNT1 is associated with altered O-glycosylation of PSA, PAP, and MUC1 in human prostate cancers.
Chen Z, Gulzar ZG, St Hill CA, Walcheck B, Brooks JD.

Abstract

BACKGROUND: 

Protein glycosylation is a common posttranslational modification and glycan structural changes have been observed in several malignancies including prostate cancer. We hypothesized that altered glycosylation could be related to differences in gene expression levels of glycoprotein synthetic enzymes between normal and malignant prostate tissues.

METHODS: 

We interrogated prostate cancer gene expression data for reproducible changes in expression of glycoprotein synthetic enzymes. Over-expression of GCNT1 was validated in prostate samples using RT-PCR. ELISA was used to measure core 2 O-linked glycan sialyl Lewis X (sLe(x) ) of prostate specific antigen (PSA), Mucin1 (MUC1), and prostatic acidic phosphatase (PAP) proteins.

RESULTS: 

A key glycosyltransferase, GCNT1, was consistently over-expressed in several prostate cancer gene expression datasets. RT-PCR confirmed increased transcript levels in cancer samples compared to normal prostate tissue in fresh-frozen prostate tissue samples. ELISA using PSA, PAP, and MUC1 capture antibodies and a specific core 2 O-linked sLe(x) detection antibody demonstrated elevation of this glycan structure in cancer compared to normal tissues for MUC1 (P = 0.01), PSA (P = 0.03) and near significant differences in PAP sLe(x) levels (P = 0.06). MUC1, PSA and PAP protein levels alone were not significantly different between paired normal and malignant prostate samples.

CONCLUSIONS: 

GCNT1 is over-expressed in prostate cancer and is associated with higher levels of core 2 O-sLe(x) in PSA, PAP and MUC1 proteins. Alterations of O-linked glycosylation could be important in prostate cancer biology and could provide a new avenue for development of prostate cancer specific glycoprotein biomarkers.



Clin Cancer Res. 2013 Jul 15;19(14):3844-55. doi: 10.1158/1078-0432.CCR-13-0505. 
Targeting natural killer cells to acute myeloid leukemia in vitro with a CD16 x 33 bispecific killer cell engager and ADAM17 inhibition.
Wiernik A, Foley B, Zhang B, Verneris MR, Warlick E, Gleason MK, Ross JA, Luo X, Weisdorf DJ, Walcheck B, Vallera DA, Miller JS.

Abstract

PURPOSE: 

The graft versus leukemia effect by natural killer (NK) cells prevents relapse following hematopoietic stem cell transplantation. We determined whether a novel bispecific killer cell engager (BiKE) signaling through CD16 and targeting CD33 could activate NK cells at high potency against acute myelogenous leukemia (AML) targets.

EXPERIMENTAL DESIGN: 

We investigated the ability of our fully humanized CD16 × CD33 (CD16 × 33) BiKE to trigger in vitro NK cell activation against HL60 (CD33(+)), RAJI (CD33(-)), and primary AML targets (de novo and refractory) to determine whether treatment with CD16 × 33 BiKE in combination with an ADAM17 inhibitor could prevent CD16 shedding (a novel inhibitory mechanism induced by NK cell activation) and overcome inhibition of class I MHC recognizing inhibitory receptors.

RESULTS: 

NK cell cytotoxicity and cytokine release were specifically triggered by the CD16 × 33 BiKE when cells were cultured with HL60 targets, CD33(+) de novo and refractory AML targets. Combination treatment with CD16 × 33 BiKE and ADAM17 inhibitor resulted in inhibition of CD16 shedding in NK cells, and enhanced NK cell activation. Treatment of NK cells from double umbilical cord blood transplant (UCBT) recipients with the CD16 × 33 BiKE resulted in activation, especially in those recipients with cytomegalovirus reactivation.

CONCLUSION: 

CD16 × 33 BiKE can overcome self-inhibitory signals and effectively elicit NK cell effector activity against AML. These in vitro studies highlight the potential of CD16 × 33 BiKE ± ADAM17 inhibition to enhance NK cell activation and specificity against CD33(+) AML, which optimally could be applied in patients with relapsed AML or for adjuvant antileukemic therapy posttransplantation.



Blood. 2013 May 2;121(18):3599-608. doi: 10.1182/blood-2012-04-425397.
NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17).
Romee RFoley B, Lenvik T, Wang Y, Zhang B, Ankarlo D, Luo X, Cooley S, Verneris M, Walcheck B, Miller J.

Abstract

The Fc receptor CD16 is present on essentially all CD56(dim) peripheral blood natural killer (NK) cells. Upon recognition of antibody-coated cells it delivers a potent signal to NK cells, which eliminate targets through direct killing and cytokine production. Here we investigated the regulation of CD16 surface expression after NK cell activation. Cytokine activation and target cell stimulation led to marked decreases in CD16 expression. Activation of CD56(dim) NK cells by cross-linking CD16 with antibodies resulted in a loss of CD16 and CD62L, which correlated with increased interferon-γ production. A disintegrin and metalloprotease-17 (ADAM17) is shown to be expressed by NK cells, and its selective inhibition abrogated CD16 and CD62L shedding, and led to enhanced interferon-γ production, especially when triggering was delivered through CD16. Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets was also enhanced by ADAM17 inhibition. This supports an important role for targeting ADAM17 to prevent CD16 shedding and improve the efficacy of therapeutic antibodies. Our findings demonstrate that over-activation of ADAM17 in NK cells may be detrimental to their effector functions by down-regulating surface expression of CD16 and CD62L.



Biochim Biophys Acta. 2013 Mar;1833(3):680-5. doi: 10.1016/j.bbamcr.2012.11.027.
ADAM17 cleaves CD16b (FcγRIIIb) in human neutrophils.
Wang Y, Wu J, Newton R, Bahaie NS, Long C, Walcheck B.

Abstract

CD16b (FcγRIIIb) is exclusively expressed by human neutrophils and binds IgG in immune complexes. Cell surface CD16b undergoes efficient ectodomain shedding upon neutrophil activation and apoptosis. Indeed, soluble CD16b is present at high levels in the plasma of healthy individuals, which appears to be maintained by the daily turnover of apoptotic neutrophils. At this time, the principal protease responsible for CD16b shedding is not known. We show that CD16b plasma levels were significantly decreased in patients administered a selective inhibitor targeting the metalloproteases ADAM10 and ADAM17. Additional analysis with inhibitors selective for ADAM10 or ADAM17 revealed that only inhibition ofADAM17 significantly blocked the cleavage of CD16b following neutrophil activation and apoptosis. CD16b shedding by ADAM17 was further demonstrated using a unique ADAM17 function-blocking mAb and a cell-based ADAM17 reconstitution assay. Unlike human CD16, however, mouse CD16 did not undergo efficient ectodomain shedding upon neutrophil stimulation or apoptosis, indicating that this mechanism cannot be modeled in normal mice. Taken together, our findings are the first to directly demonstrate that ADAM17 cleaves CD16 in human leukocytes.





J Leukoc Biol. 2012 Sep;92(3):667-72. doi: 10.1189/jlb.0312112.
ADAM17 activation in circulating neutrophils following bacterial challenge impairs their recruitment.
Long C, Hosseinkhani MR, Wang Y, Sriramarao P, Walcheck B.

Abstract

Neutrophil infiltration and bacterial clearance occur earlier in conditional knockout mice with leukocytes lacking the metalloprotease ADAM17 than in control mice. We investigated cell-intrinsic changes in neutrophils lacking ADAM17 and alterations in the inflammatory environment in conditional ADAM17 knockout mice to determine how the sheddase exerts its effects on neutrophil recruitment. In vivo analyses comparing control and ADAM17-deficient neutrophils revealed that the latter cells accumulated at increased levels in the inflamed mesenteric microvasculature and in the peritoneal cavity following bacterial challenge, indicating changes in their adhesive properties. Consistent with this, bacterial infection caused a marked down-regulation of L-selectin, an adhesion protein and substrate of ADAM17, from the surface of circulating neutrophils in control mice but not in conditional ADAM17 knockout mice. Neutrophils from gene-targeted mice with leukocytes expressing a noncleavable form of L-selectin also displayed a competitive advantage in the presence of control neutrophils when infiltrating a site of infection. Taken together, our findings reveal that impaired L-selectin shedding is a key mechanism underlying early neutrophil recruitment in conditional ADAM17 knockout mice during bacterial infection. Disrupting only the shedding of L-selectin, however, did not increase bacterial clearance, indicating that additional substrates also contribute to the detrimental role of ADAM17 during severe infection.