Distinct Roles for Neutrophils and Dendritic Cells
in Inflammation and Autoimmunity in motheaten Mice
Clare L. Abram,1Gray L. Roberge,1Lily I. Pao,2,3Benjamin G. Neel,2,4,* and Clifford A. Lowell1,*
1Department of Laboratory Medicineandthe Programin Immunology, University of California,San Francisco, San Francisco, CA 94143, USA
MA 02215, USA
3Present address: Five Prime Therapeutics, Inc., South San Francisco, CA 94080, USA
4Present address: Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health
Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario MG5 1L7, Canada
*Correspondence: email@example.com (B.G.N.), firstname.lastname@example.org (C.A.L.)
for complex autoimmune and inflammatory disease.
Null mutations in Ptpn6, which encodes the nonre-
ceptor protein-tyrosine phosphatase Shp1, cause
the motheaten phenotype. However, Shp1 regulates
multiple signaling pathways in different hematopoi-
etic cell types, so the cellular and molecular mecha-
nism of autoimmunity and inflammation in the
motheaten mouse has remained unclear. By using
floxed Ptpn6 mice, we dissected the contribution of
innate immune cells to the motheaten phenotype.
Ptpn6 deletion in neutrophils resulted in cutaneous
inflammation, but not autoimmunity, providing an
animal model of human neutrophilic dermatoses.
By contrast, dendritic cell deletion caused severe
autoimmunity, without inflammation. Genetic and
biochemical analysis showed that inflammation was
caused by enhanced neutrophil integrin signaling
through Src-family and Syk kinases, whereas auto-
immunity resulted from exaggerated MyD88-depen-
dent signaling in dendritic cells. Our data demon-
strate that disruption of distinct Shp1-regulated
pathways in different cell types combine to cause
Shultz in 1975 as a spontaneous autosomal recessive mutation
leading to immune deficiency, widespread inflammation, and
death at 2 to 4 weeks of age (Green and Shultz, 1975). Later,
a related mutant strain was discovered (motheaten viable, or
mev/mev) in which the homozygous mice display a similar,
though less severe, phenotype and survive for several more
(9–12) weeks (Shultz et al., 1984). Both lines of mice exhibit
chronic inflammation of the skin (leading to fur loss, hence the
name ‘‘motheaten’’), accompanied by the production of autoan-
tibodies and immune complex deposition in tissues. me/me and
mev/mevmice also develop a lethal pneumonitis characterized
by neutrophil and macrophage accumulation in the lungs
(Jiao et al., 1997). Gene-mapping studies show that distinct
mutations in Ptpn6, which encodes the protein-tyrosine phos-
phatase Shp1, cause the me/me and mev/mevphenotypes.
The Ptpn6me/memutation results in complete loss of Shp1
protein, whereas Ptpn6me-v/me-vmice express wild-type (WT)
amounts of two mutant forms of Shp1 with greatly reduced cata-
lytic activity (Shultz et al., 1997). Recently, two strains of mice
with new Ptpn6 point mutations were reported, Ptpn6spin/spin
and Ptpn6meB2/meB2, both of which have milder phenotypes
than seen in Ptpn6me-v/me-vmice (Croker et al., 2008; Nestero-
vitch et al., 2011a). The motheaten phenotype has long served
as a paradigm for a complex autoimmune and inflammatory
disease; therefore, elucidating its pathogenesis is of general
Substantial effort has been devoted to investigating the func-
tion of Shp1 in the immune system (Pao et al., 2007a; Tsui et al.,
2006). Nevertheless, its detailed role in WT mice, and an under-
standing of how Ptpn6 mutations cause autoimmunity and
inflammation, remains unclear. All hematopoietic cells express
Shp1, and their complicated interactions have made it difficult
to dissect the relative contributions of different cell types to mo-
theaten disease. Transplantation experiments indicate that the
motheaten phenotype is due predominantly to bone-marrow-
derived cells. Moreover, pretreatment of motheaten mice with
anti-CD11b prevents the development of skin inflammation,
pneumonitis, splenomegaly, and defective T cell function (Koo
et al., 1993). These data indicate that myeloid cells not only
cause the inflammatory disease but also influence the develop-
ment of autoimmunity in motheaten mice. Consistent with this
conclusion, analysis of Ptpn6me-v/me-vmice on a Rag1-deficient
background shows that B and T lymphocytes are dispensable
for the inflammatory disease (Yu et al., 1996).
In addition to acting in several hematological cell types,
Shp1 is implicated in the regulation of multiple signaling path-
kinases, G protein coupled receptors, Toll-like receptors (TLRs),
and cytokine receptors (Neel et al., 2003; Pao et al., 2007a;
Zhang et al., 2000). Much previous work has focused on study-
ing cells isolated from mice containing Ptpn6 mutations. For
example, neutrophils from Ptpn6me/meand Ptpn6me-v/me-vmice
are hyperadhesive and show defective chemotaxis, perhaps
Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc. 489
Figure 1. Disease Phenotype in Ptpn6fl/flS100a8-cre and Ptpn6fl/flItgax-cre Mice
(A and C) Mice of the indicated genotypes were monitored every two (Ptpn6fl/flS100a8-cre) or four (Ptpn6fl/flItgax-cre) weeks for paw inflammation or lymph-
adenopathy, respectively. The presence of these phenotypes was scored, and the percentage of ‘‘disease-free’’ mice at each time point was graphed using
Prism, ***p < 0.001.
(legend continued on next page)
Deconvoluting the Phenotype of motheaten Mice
490 Immunity 38, 489–501, March 21, 2013 ª2013 Elsevier Inc.
Detection of Serum Anti-Nuclear Antibodies and Cytokines
Blood was collected in Z-gel microtubes (Sarstedt) and left to clot at RT for
30–45 min. Then, tubes were centrifuged at 6,000 3 g for 2 min to obtain
serum. Sera were diluted 1:40 and applied to Kallestad HEp-2 slides (Bio-
Rad). Anti-nuclear antibodies were detected using FITC conjugated goat
tion, Adobe Photoshop was used to determine mean pixel intensity in the
green channel. For anti-dsDNA Ig ELISA, 96-well flat-bottom polystyrene
plates (Microtest 351172, BD) were coated with 20 ng/well of linearized
pUC19 plasmid in 100 mM Tris-HCl. After overnight incubation at room
temperature, plates were blocked with PBS containing 2% (vol/vol) FCS and
0.05% (vol/vol) Tween 20 for 30 min. Sera, diluted 1:40–1:600, were added
to the plate and incubated for 2 hr at room temperature. The assays were
developed with horseradish-peroxidase-conjugated goat anti-mouse IgG
(1:3,000) or IgM (1:5,000) (Bethyl Laboratories). After addition of TMB Micro-
well Peroxidase Substrate System (KPL), reactions were terminated with
1 M phosphoric acid (Sigma-Aldrich), and the absorbance at 450 nm (A450)
was measured by using a Spectra Max Plus microplate reader (Molecular
Cytokineand chemokine concentrations
quantified using a Milliplex?MAP mouse cytokine and chemokine kit from
Millipore, according to the manufacturer’s instructions. The concentration of
TNF-a in BMDC culture supernatants was determined by ELISA (R&D
in mousesera were
Cellular Functional Assays and Biochemical Analysis
Neutrophils, purified from bone marrow as described previously (Mo ´csai
et al., 2002, 2006) or by using a five-step gradient (Siemsen et al., 2007),
were stimulated as described (Mo ´csai et al., 2002, 2006). Adhesion-depen-
dent respiratory burst was measured as reported previously (Mo ´csai et al.,
2002) or by using isoluminol-enhanced chemiluminescence (Dahlgren
et al., 2007). Bone-marrow-derived macrophages were differentiated
in vitro from mononuclear cells as described (Mo ´csai et al., 2006). BMDCs
were differentiated in vitro from mononuclear cells cultured for 11 days in
medium containing GM-CSF. Cells were serum-starved for 12 hr prior to
stimulation. Bone-marrow mononuclear cells were spin-infected with retro-
virus as described (Mo ´csai et al., 2006), followed by differentiation in medium
disease-free mice was plotted by using Kaplan-Meir survival analysis and
analyzed by using a log-rank (Mantel-Cox) test. Differences between two
groups were assessed by the unpaired t test; differences between three or
more groups were evaluated by ANOVA, followed by Bonferroni’s Multiple
Comparison post-test. Differences between observed and expected allele
when p % 0.05.
Supplemental Information includes five figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
We thank Yongmei Hu for help with animal husbandry and genotyping;
and Chrystelle Lamagna for help with FACS sorting. Supported by the US
National Institutes of Health (AI065495, AI068150 and AI078869 to C.A.L.,
RO1CA114945 and R37CA49152 to B.G.N.). B.G.N. is a Canada Research
Chair, Tier I, and is partially supported by the Ontario Ministry of Health and
Long Term Care and the Princess Margaret Hospital Foundation.
Received: May 4, 2012
Accepted: November 26, 2012
Published: March 21, 2013
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