Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0307193 on May 17, 2007

Published online before print May 17, 2007
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(Journal of Leukocyte Biology. 2007;82:173-176.)
© 2007 by Society for Leukocyte Biology

Role of ADAM17 in the ectodomain shedding of TNF-{alpha} and its receptors by neutrophils and macrophages

Jessica H. Bell, Amy H. Herrera, Ying Li and Bruce Walcheck1

Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, USA

1 Correspondence: University of Minnesota, 295j AS/VM Bldg., 1988 Fitch Avenue, St. Paul, MN 55108, USA. E-mail: walch003{at}umn.edu

ABSTRACT

TNF-{alpha} and its receptors TNFRI and TNFRII are cleaved from the surface of leukocytes by a proteolytic process referred to as ectodomain shedding. The role of a disintegrin and metalloproteinase 17 (ADAM17) in this process by the major professional phagocytes neutrophils and macrophages, the primary producers of TNF-{alpha} during inflammation induction, is based entirely on indirect evidence, and other sheddases have been implicated as well. As Adam17 gene-targeting in mice is lethal, we assessed the protease’s relative contribution to TNF-{alpha}, TNFRI, and TNFRII shedding using radiation chimeric mice with leukocytes lacking functional ADAM17. We report ablated, soluble TNF-{alpha}, TNFRI, and TNFRII production by neutrophils and macrophages stimulated with various microbial antigens and greatly reduced TNF-{alpha} levels in vivo following inflammation induction. This is the first simultaneous analysis of TNF-{alpha}, TNFRI, and TNFRII shedding by neutrophils and macrophages and the first direct evidence that ADAM17 is a primary and nonredundant sheddase.

Key Words: inflammation • metalloprotease

The cytokine TNF-{alpha} induces an extensive array of pro- and anti-inflammatory effects [1 ] and is primarily produced by neutrophils and macrophages upon inflammation induction [2 ]. TNF-{alpha} is synthesized as a trimeric type II transmembrane precursor, which is converted to a soluble form by a proteolytic process referred to as ectodomain shedding [3 ]. There are two cell surface receptors for TNF-{alpha}, TNFR type I (TNFRI) and TNFR type II (TNFRII) [1 ]. Like TNF-{alpha}, these receptors also undergo ectodomain shedding, generating soluble, functional receptors [3 ].

Ectodomain shedding by leukocytes is mediated primarily by serine proteases and disintegrin metalloproteases [3 ]. A disintegrin and metalloproteinase 17 (ADAM17) has been reported to be a sheddase of TNF-{alpha} and its receptors in various cell culture systems [4 5 6 7 ]. At this time, however, there is no direct evidence that ADAM17 is the primary sheddase of TNF-{alpha}, TNFRI, and TNFRII in neutrophils and macrophages. Moreover, a variety of in vitro and in vivo studies has demonstrated that sheddases other than ADAM17 can cleave TNF-{alpha}, including matrix metalloprotease-7 [8 ], ADAM9, -10, and -19 [9 10 11 12 13 ], and the serine protease proteinase 3 [14 ]. Sheddases other than ADAM17 cleave the TNFRs as well, such as the serine protease elastase [15 ]. Taken together, there is substantial evidence for multiple sheddase activities being involved in the cleavage of TNF-{alpha}, TNFRI, and TNFRII.

Determining the contribution of ADAM17 in the shedding of TNF-{alpha} and its receptors by primary, mature neutrophils and macrophages has been hindered by the lethality of targeting the Adam17 gene [6 ]. Therefore, to address this important question, we used radiation chimeric mice reconstituted with blood cells deficient in functional ADAM17.

ADAM17 DEFICIENCY IN MACROPHAGES AND NEUTROPHILS ABLATES SOLUBLE TNF-{alpha} PRODUCTION

We previously reported the generation of radiation chimeric mice reconstituted with leukocytes deficient in functional ADAM17 [16 ]. For irradiated mice reconstituted with wild-type or ADAM17-deficient leukocytes (referred to here as wild-type or ADAM17 chimeric mice), the percentage of chimerism was >95%, as determined by the expression of the congenic marker CD45.2 (data not shown). ADAM17 deficiency by leukocytes from ADAM17 chimeric mice was verified by their lack of inducible L-selectin shedding [16 ].

Bone marrow neutrophils were isolated from age-matched, wild-type, and ADAM17 chimeric mice. Equivalent numbers of cells from both groups of chimeric mice were stimulated through TLR4 by LPS or by phagocytosis of Escherichia coli bioparticles or Staphylococcus aureus bioparticles, and then, the cell supernatants were examined for soluble TNF-{alpha} levels by ELISA. Neutrophils from the ADAM17 chimeric mice were found to produce greatly reduced levels of soluble TNF-{alpha} compared with neutrophils from wild-type, chimeric mice (Fig. 1A ). Neutrophils from both groups of chimeric mice, however, underwent similar levels of activation [16 ], and their production of cell-associated TNF-{alpha} was similar before and after activation (Fig. 1A) , indicating that cell activation as well as TNF-{alpha} synthesis were not affected by the absence of functional ADAM17. In addition, a disruption in de novo TNF-{alpha} synthesis has not been reported in studies involving ADAM17-deficient cell lines or cells treated with metalloprotease inhibitors [4 , 6 , 7 , 13 , 17 ]. Peritoneal exudate macrophages from wild-type and ADAM17 chimeric mice were activated and examined in the same manner. Supernatants from macrophages deficient in functional ADAM17 had essentially undetectable levels of soluble TNF-{alpha} as well (Fig. 1B) .


Figure 1
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  		Figure 1. Soluble TNF-{alpha} production by macrophages and neutrophils from wild-type and ADAM17 chimeric mice. Radiation chimeric mice were generated as described previously [16 ]. Briefly, irradiated B6.SJL (CD45.1) mice were injected with fetal liver cells isolated from ADAM17+/+ or ADAM17{Delta}Zn/{Delta}Zn embryos [C57BL/6J/129Sv (CD45.2)], which are referred to here as wild-type or ADAM17 chimeric mice. Bone marrow neutrophils were isolated as described previously [16 ], and peritoneal macrophages were isolated 96 h after injection of mice with thioglycollate (1 ml 3% w/v). (A) Bone marrow neutrophils and (B) peritoneal exudate macrophages (2x106) from the wild-type or ADAM17 chimeric mice, as indicated, were cultured for 6 h in the presence of LPS (List Biological Laboratories, Campbell, CA, USA; isolated from E. coli K-12 LCD25, 10 µg/ml), E. coli bioparticles, or S. aureus bioparticles (Molecular Probes, Eugene, OR, USA; cells:bioparticles=1:10). Neutrophils were also detergent-extracted before or after LPS stimulation (A, right panel). For each experiment, cells were plated in triplicate wells. (C) Peritoneal fluid and (D) blood plasma from the wild-type and ADAM17 chimeric mice were collected 90 min after i.p. injection of LPS (400 µg in 1 ml PBS). n.d., Not detected. Supernatant, detergent lysate, peritoneal fluid, and plasma levels of TNF-{alpha} were determined by ELISA (R&D Systems, Minneapolis, MN, USA). All samples were filtered through a 0.22-µm syringe filter to remove cell debris prior to ELISA analysis. Note that for Panels A and B, TNF-{alpha} levels are in pg/ml, whereas for Panels C and D, TNF-{alpha} levels are in ng/ml. Data represent the mean ± SD of more than or equal to three independent experiments.

 
The treatment of normal mice with a bolus LPS injection produces an acute and synchronized, inflammatory response, which results in transient TNF-{alpha} levels in the blood [18 ]. Following i.p. administration of LPS into wild-type and ADAM17 chimeric mice, local and systemic production of soluble TNF-{alpha} by the latter group of chimeric mice was reduced severely (Fig. 1C and 1D) . Peritoneal cavity and peripheral blood leukocyte counts and differentials for untreated and LPS-injected, wild-type, and ADAM17 chimeric mice were not significantly different (data not shown and ref. [16 ]), suggesting that the dissimilar levels of TNF-{alpha} in the peritoneal fluid and plasma from the two cohorts of chimeric mice were not a result of vastly different leukocyte numbers or proportions.

DISRUPTION OF TNFRI AND TNFRII SHEDDING BY MACROPHAGES AND NEUTROPHILS LACKING FUNCTIONAL ADAM17

Leukocytes from wild-type and ADAM17 chimeric mice were stimulated with LPS, E. coli, or S. aureus, and their production of soluble TNFRI and TNFRII was determined by ELISA. As with TNF-{alpha}, the levels of TNFRI and TNFRII in the supernatants of activated macrophages and neutrophils lacking functional ADAM17 were reduced greatly compared with cells derived from the wild-type, chimeric mice (Fig. 2A and 2B ).


Figure 2
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  		Figure 2. Soluble TNFRI and TNFRII production by macrophages and neutrophils from wild-type and ADAM17 chimeric mice. (A) Peritoneal exudate macrophages and (B) bone marrow neutrophils (2x106) from wild-type or ADAM17 chimeric mice were cultured for 6 h in the presence or absence of the indicated stimulant (x-axis). The presence of soluble TNFRI or TNFRII, as indicated (y-axis), in the cell supernatants was determined by ELISA analysis (R&D Systems), as described in Figure 1 . (C) Resting bone marrow neutrophils from wild-type (WT) and ADAM17 chimeric mice were double-stained for surface expression of TNFRI (left panel) or TNFRII (right panel; BioLegend, San Diego, CA, USA; Clones 55R-286 and TR75-89, respectively) and the neutrophil marker Gr-1 (eBiosciences, San Diego, CA, USA; Clone RB6-8C5). Relative staining levels were determined by flow cytometry (Becton Dickinson, San Jose, CA, USA; FACSCanto). The dashed lines indicate staining by an appropriate, isotype-matched, negative control mAb (Caltag, S. San Francisco, CA, USA). Results are representative of greater than or equal to four independent experiments.

 
Of interest was that resting neutrophils from ADAM17 chimeric mice expressed higher levels of surface TNFRI and TNFRII compared with resting neutrophils from wild-type, chimeric mice, as determined by flow cytometry (Fig. 2C) , suggesting that ADAM17 participates in regulating the homeostatic density of TNFRI and TNFRII as well. Noncleavable, cell surface determinants on neutrophils, such as membrane-activated complex-1 and myeloid differentiation antigen Gr-1, were expressed at equivalent levels on resting neutrophils from wild-type and ADAM17 chimeric mice (data not shown and ref. [16 ]), demonstrating that ADAM17 deficiency did not cause a global up-regulation in expression of cell surface molecules. We have not observed a consistent increase in the expression level of TNF-{alpha} by ADAM17-deficient leukocytes (Fig. 1 and data not shown). It is interesting that Ruuls et al. [19 ] reported the same thing for leukocytes from gene-targeted mice expressing noncleavable TNF-{alpha}, and they proposed that membrane TNF-{alpha} levels may be regulated by a feedback mechanism independent of shedding.

In summary, we demonstrate for the first time that ADAM17 plays a primary role in TNF-{alpha}, TNFRI, and TNFRII shedding by mature neutrophils and macrophages stimulated by LPS and phagocytosis and upon inflammation induction in vivo. In most of our assays, however, ADAM17 deficiency did not result in a complete abrogation of shedding, which may be a result, in part, of low-level cleavage by additional sheddase mechanisms. This is consistent with other sheddases being implicated in the shedding of TNF-{alpha}, TNFRI, and TNFRII, as described above.

ACKNOWLEDGEMENTS

Funding for these studies was provided by the National Institutes of Health (HL61613). We thank Drs. David Lee, Susan Sunnarborg, Roy Black, and Jacques Peschon for providing a breeding pair of ADAM17+/{Delta}Zn mice and Elaine Raines for helpful discussions.

Received March 28, 2007; revised April 30, 2007; accepted May 4, 2007.

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