of July 25, 2016.This information is current as

Autoimmune Disease DevelopmentRegulators of B Cell Receptor Signaling in Genetic Interdependence of Lyn and Negative

HibbsKenneth W. Harder, David M. Tarlinton and Margaret L. Evelyn Tsantikos, Mhairi J. Maxwell, Nicole Kountouri,

http://www.jimmunol.org/content/189/4/1726doi: 10.4049/jimmunol.1103427July 2012;

2012; 189:1726-1736; Prepublished online 13J Immunol 

Referenceshttp://www.jimmunol.org/content/189/4/1726.full#ref-list-1

, 23 of which you can access for free at: cites 52 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on July 25, 2016

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Genetic Interdependence of Lyn and Negative Regulators ofB Cell Receptor Signaling in Autoimmune DiseaseDevelopment

Evelyn Tsantikos,*,†,‡ Mhairi J. Maxwell,*,† Nicole Kountouri,† Kenneth W. Harder,†,1

David M. Tarlinton,x and Margaret L. Hibbs*,†

Ab-mediated autoimmune disease is multifaceted and may involve many susceptibility loci. The majority of autoimmune patients

are thought to have polymorphisms in a number of genes that interact in different combinations to contribute to disease patho-

genesis. Studies in mice and humans have implicated the Lyn protein tyrosine kinase as a regulator of Ab-mediated autoimmune

disease. To examine whether haploinsufficiency of Lyn gives rise to cellular and clinical manifestations of autoimmune disease, we

evaluated the phenotype of Lyn+/2 mice. We find that their B cell compartment is significantly perturbed, with reduced numbers

of marginal zone and transitional stage 2 B cells, expansion of plasma cells, downregulation of surface IgM, and upregulation of

costimulatory molecules. Biochemical studies show that Lyn+/2 B cells have defects in negative regulation of signaling, whereas

Lyn+/2 mice develop IgG autoantibodies and glomerulonephritis with age. Because Lyn has a pivotal role in the activation of

inhibitory phosphatases, we generated mice harboring double heterozygous loss-of-function mutations in Lyn and SHP-1 or Lyn

and SHIP-1. Partial inactivation of SHP-1 or SHIP-1 amplifies the consequence of Lyn haploinsufficiency, leading to an accelerated

development of autoantibodies and disease. Our data also reveal that the BALB/c background is protective against autoimmune-

mediated glomerulonephritis, even in the face of high titer autoantibodies, whereas the C57BL/6 background is susceptible. This study

demonstrates that Lyn is a haploinsufficient gene in autoimmune disease and importantly shows that quantitative genetic variation

in Lyn-regulated pathways can mirror the complete loss of a single critical inhibitory molecule. The Journal of Immunology, 2012, 189:

1726–1736.

Human diseases are rarely caused by a single geneticmutation. For instance, systemic lupus erythematosus(SLE) is a complex polygenic autoimmune disease that

may involve up to 100 susceptibility genes, and it is likely thatpolymorphisms in different genes with distinct combinationscontribute to SLE pathogenesis (1). In mice, several homozygousgenetic mutations mimic, to varying degrees, the pathogenesis ofSLE, suggesting that pathways regulated by the missing or over-active gene products in mice are similarly affected in humans. Forexample, augmented CD19 signaling, which triggers autoim-mune disease in mice (2), is implicated in systemic sclerosis (3),

whereas elevated levels of circulating BAFF, which lead to auto-immunity in mice (4), are seen in patients with Sjogren’s syn-drome (5). Furthermore, it is known that mutations in thecomplement pathway in mice and humans are risk factors for SLE(reviewed in Ref. 6). However, for the most part, human patientsare less likely to suffer from the overexpression or complete ab-sence of just one regulatory factor but, rather, are anticipated toshow quantitative genetic variation in several factors along a bio-chemical pathway.Lyn tyrosine kinase is an important regulator of Ab-mediated

autoimmune disease in mice and humans (reviewed in Ref. 7).Both Lyn-deficient and Lyn gain-of-function mutant mice developcirculating autoreactive Abs and severe glomerulonephritis causedby the deposition of immune complexes in the kidney, a pathologyreminiscent of SLE (8–10). Early studies of human SLE patientsshowed some to have abnormal levels of Lyn (11, 12), whereasmore recent data suggested that Lyn expression and localization tolipid raft-signaling domains were altered in a majority of patientswith SLE (13). Moreover, genome-wide association scans andpopulation-based studies identified Lyn as an SLE susceptibilitylocus, with single nucleotide polymorphisms being found in the59 untranslated region of Lyn (14, 15). Collectively, these studiesimply that alteration of Lyn activity or expression may be asso-ciated with human disease.Although Lyn has long been known to be an activatory com-

ponent of the BCR-signaling complex, it is now clear that Lyn isalso an essential inhibitory-signaling molecule downstream of theBCR (7). Lyn activity is required for the tyrosine phosphorylationof inhibitory cell surface receptors, such as FcgRIIB1, CD22, andPIR-B, generating binding sites for negative regulatory phospha-tases, such as the 59 inositol phosphatase SHIP-1 and the protein

*Leukocyte Signaling Laboratory, Department of Immunology, Monash University,Central Clinical School, Alfred Medical Research and Education Precinct, Mel-bourne, Victoria 3004, Australia; †Ludwig Institute for Cancer Research, Parkville,Victoria 3050, Australia; ‡Department of Surgery, University of Melbourne,Parkville, Victoria 3010, Australia; and xWalter and Eliza Hall Institute of MedicalResearch, Parkville, Victoria 3052, Australia

1Current address: Department of Microbiology and Immunology, Life Sciences Cen-tre, Vancouver, British Columbia, Canada.

Received for publication November 29, 2011. Accepted for publication June 1, 2012.

This work was supported in part by grants from the National Health and MedicalResearch Council of Australia. E.T. is supported by an Australian PostgraduateAward from the Australian Government, and M.L.H., M.J.M., and D.M.T. are recip-ients of fellowships from the National Health and Medical Research Council ofAustralia.

Address correspondence and reprint requests to Dr. Margaret L. Hibbs, LeukocyteSignaling Laboratory, Department of Immunology, Monash University, Central Clin-ical School, Alfred Medical Research and Education Precinct, Commercial Road,Melbourne, Victoria 3004, Australia. E-mail address: Margaret.Hibbs@monash.edu

Abbreviations used in this article: ANA, anti-nuclear Ab; MZB, marginal zoneB cell; SLE, systemic lupus erythematosus; T2, transitional stage 2.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103427

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

tyrosine phosphatase SHP-1 (16-21). Consistent with the impor-tance of inhibitory signaling pathways for limiting cellular acti-vation, loss-of-function mutations in these regulatory moleculesinvoke immune system defects. Loss of SHIP-1 leads to deregu-lated signaling in the PI3K pathway, which, in turn, results inmyeloproliferative disease in mice (22, 23), perturbations in B celldevelopment and function (24–26), and an SLE-like autoimmunedisease that is B cell dependent (27). The failure to appropriatelyrecruit and/or activate SHP-1 leads to catastrophic deregulation ofthe immune system, as evidenced by the severe phenotypes ofmice carrying loss-of-function mutations in SHP-1 (Me, Mev,spin), which include systemic autoimmunity (28–30). Loss ofCD22, a site of membrane recruitment for SHP-1, leads to a mildphenotype, dependent on genetic background synergizing with theeffects of CD22 deficiency to promote autoantibody development(31–34). Although there is some ambiguity about the impor-tance of CD22 in autoimmune disease, it is clear that loss ofCD22 function is associated with a hyperresponsive B cell phe-notype. In studies exploring the CD22, Lyn, SHP-1–signalingpathway, haploinsufficiency of Lyn, CD22, and SHP-1 in triple-heterozygous CD22+/2Lyn+/2Mev+/2 mice led to reduced CD21and IgM expression and increased MHC class II expression onmature B cells compared with single- and double-heterozygousmice, whereas the mice showed increased serum IgM and in-creased negative selection in the presence of a neo self-Ag (17).Whether these B cell changes are the extent of the consequencesof this multilocus heterozygosity or, more importantly, whetherthey result in autoimmunity, has not been determined. In morerecent studies, we found that haploinsufficiency of Lyn in miceleads to the development of autoantibodies, albeit with delayedonset compared with Lyn2/2 mice; however, we did not assessdisease development (35).Both mouse and human studies suggest that Lyn-dependent

pathways contribute to the development of a subset of autoim-mune conditions that, in humans, includes SLE. However, patientsare unlikely to possess single genetic changes that confer consti-tutive Lyn activity or inactivity; rather, it seems more probable thatthey have perturbations in steps along Lyn-regulated pathways thatcollectively have an effect similar to that observed in mice. Tomimic this type of situation, we investigated the consequence ofhaploinsufficiency of Lyn, either alone or in combination withSHP-1 or SHIP-1. We establish that Lyn is a haploinsufficient genein autoimmune disease and demonstrate that simultaneous hap-loinsufficiency of SHP-1 or SHIP-1 greatly accelerates autoim-mune disease development. These types of genetic interactionsmore appropriately reflect the human condition, where suscepti-bility to, and manifestation of, autoimmune disease is more likelyto be dependent on the accumulation of effects on multiple genesrather than the single complete loss of a particular gene.

Materials and MethodsAnimals

Lyn2/2 mice have been described (8), and mice that had been backcrossedfor 20 generations to the C57BL/6 background were used. Motheaten vi-able (Mev) mice (29, 36) were on a C57BL/6 background and weremaintained by interbreeding heterozygotes and genotyping. HeterozygoteLyn+/2 animals were generated by crossing Lyn2/2 mice with C57BL/6mice; double-heterozygote Lyn+/2Mev+/2 mice were generated by inter-breeding Lyn2/2 and Mev+/2 mice and genotyping. To generate compoundmutants involving Lyn and SHIP-1, both C57BL/6 and BALB/c geneticbackground mice were used, which were described previously (27, 35, 37,38). Lyn+/2SHIP-1+/2 mice were generated by interbreeding male SHIP-12/2 mice with female Lyn2/2 mice. All experiments were performedin accordance with National Health and Medical Research Council ofAustralia guidelines for the care and use of animals for scientific purposes

and were approved by the Animal Ethics Committees of the Ludwig In-stitute for Cancer Research/Department of Surgery and the Baker Heartand International Diabetes Institute, Melbourne.

Cell preparation and flow cytometry

Single-cell suspensions of spleen were prepared by extruding the cells fromthe capsule and then passing through a 40-mm filter. Multicolor flowcytometry was used to examine lymphoid tissue composition, as described(35).

Proliferation assay

Spleen cells were isolated from 10-wk-oldmice, and red cells were removedby lysis with Tris-buffered ammonium chloride. Single-cell suspensionswere incubated with biotinylated Abs to CD5, CD43, Mac-1, CD11c, Ter-119, and Gr-1 (BD Biosciences Pharmingen, San Diego, CA), and non-B cells were depleted using Streptavidin MicroBeads and MiniMACScolumns (Miltenyi Biotec, North Ryde, NSW, Australia). Purified cells wereresuspended in DMEM, 5% FCS, 100 U/ml penicillin-streptomycin, and 4mM glutamine and then cultured for 3 d with no stimulus, 10 mg/ml F(ab9)2goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, WestGrove, PA), 10 mg/ml intact goat anti-mouse IgM (Jackson Immuno-Research Laboratories), or 5 mg/ml LPS (Sigma). On day 3, cells werepulsed with [3H]thymidine for 6 h prior to harvesting and counting witha b-scintillation counter (Perkin Elmer, Wellesley, MA).

SDS-PAGE and Western blot

For signaling studies, B cells were purified from spleen, as outlined for theproliferation assay. B cells were stimulated with 20 mg/ml intact goat anti-mouse IgM for 0, 5, or 30 min and then lysed at a concentration of 108

cells/ml in 1% Triton X-100, 0.1% SDS, 50 mM Tris (pH 7.5), 150 mMNaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride,and complete protease inhibitor mixture (Roche Diagnostics, Indianapolis,IN). Following protein determination by BCA (Pierce Chemical Company,Rockford, IL), total-cell lysates were separated by SDS-PAGE, and pro-teins were transferred to nitrocellulose. Blots were probed with anti–p-Erk and anti-Erk (Cell Signaling Technology, Danvers, MA) as a loadingcontrol and were detected using the Odyssey detection system (LI-CORBiosciences, Lincoln, NE).

Detection of autoantibodies by ELISA

Mice were bled from the tail vein every 2–4 wk from the age of 10 wk, andanti-nuclear Abs (ANAs) were measured, as described (35). IgG ANAvalues are defined as the optical density reading measured at 450 nm withcorrection at 595 nm.

Myeloid colony assays

Myeloid progenitor cells responsive to defined growth factors were enu-merated by semisolid agar cultures, as described (39).

Histopathology

Tissues for light microscopy were fixed in Bouin’s solution for 24 h andthen embedded in paraffin. For histopathology studies, 3-mm sections werecut and stained with H&E, according to standard procedures. Pathologicalassessment was performed by determination of glomerular area as follows.Serial photographs were taken of H&E-stained kidney sections. ImageJsoftware was used to determine the cross-sectional area of individualglomeruli from the images obtained using a known reference scale. Be-tween 20 and 30 glomeruli were analyzed/mouse, and the mean glomerulararea was determined, as described (40). For each genotype, the meanglomerular area of individual mice was plotted to determine the meanvalue for each genotype.

Statistical analysis

Statistical analysis was performed on all numerical data using GraphPadPrism software (GraphPad, San Diego, CA).

ResultsPerturbation of the B cell compartment in Lyn+/2 mice

Previous studies by us and other investigators found severe per-turbations in the B cell compartment of Lyn2/2 mice, whichprecede autoimmune disease development (8, 9, 41). To assesswhether the loss of one allele of Lyn affected this lineage, we

The Journal of Immunology 1727

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

examined the B cell compartment of Lyn+/2 mice by flowcytometry, assessing the expression of cell surface markers in-dicative of developmental and activation status (Fig. 1). Unlike inLyn2/2 mice (38), the total number of splenic B cells (Fig. 1A)did not differ between control and Lyn+/2 mice. However, similarto Lyn2/2 mice, plasma cell numbers were significantly elevatedin the spleens of Lyn+/2 mice (0.82 6 0.14 3 106 in C57BL/6versus 2.53 6 0.45 3 106 plasma cells in Lyn+/2 spleen; Fig. 1A).Lyn2/2 mice also show a total loss of the marginal zone B cell(MZB) compartment and a block in development from transitionalstage 1 to transitional stage 2 (T2) B cells (38, 42). We determinedthe effect of Lyn heterozygosity on these populations and foundthat both the MZB and T2 cell compartments were reduced toapproximately half of the number of cells present in controlspleens (Fig. 1B). Thus, loss of one allele of Lyn is sufficient toaffect the plasma cell, MZB, and T2 populations.We next characterized the activation status of B cells from

Lyn+/2 mice and found the amount of IgM on the cell surface tobe significantly downregulated compared with controls. Curiously,the fold reduction in IgM on Lyn+/2 B cells was greater thanobserved for Lyn2/2 B cells (Fig. 1A, 1C). This could be a re-flection of the unusual B cell populations present in Lyn2/2 mice(38). Although the amounts of MHC class II and CD86 on Lyn+/2

B cells were significantly elevated over those on control B cells,their expression was not as high as on Lyn2/2 B cells (Fig. 1C).To determine whether the activated phenotype of Lyn+/2 B cells

was associated with enhanced B cell responses, we first measured

proliferation of Lyn+/2 B cells after exposure to a variety ofagonists, including BCR-specific and -independent stimuli (Fig.1D). Although Lyn+/2 B cells responded like control B cells toLPS, they were mildly hyperresponsive to BCR cross-linking.Interestingly, they displayed marked hyperresponsiveness tostimulation with intact IgM Ab, which normally provides an in-hibitory signal to B cells through coligation of the BCR and in-hibitory FcgR. This suggests that this Lyn-dependent inhibitorypathway in B cells is faulty when Lyn expression is reduced. Toexamine whether this response had a biochemical corollary,B cells were stimulated with intact IgM Ab, and phosphorylationof Erk was assessed by probing with anti–p-Erk Ab, followedby anti-Erk as a loading control (Fig. 1E). In control and Lyn+/2

B cells, phosphorylation of Erk was maximal at 5 min poststim-ulation; however, the signal was more intense in Lyn+/2 B cells, asdetermined by densitometry (Fig. 1E). At 30 min poststimulation,when the intensity of p-Erk in control B cells had largely di-minished to baseline levels, the signal was sustained in Lyn+/2

B cells.

T cell activation occurs in the absence of early myeloid celldefects in Lyn+/2 mice

We previously showed that although Lyn is not expressed in T cells,T cell perturbations occur in aged Lyn2/2 mice (35, 38). Weconcluded that this was due to the effects of the inflammatoryenvironment and other cellular compartments in Lyn2/2 mice onT cells. Therefore, we were interested to determine whether ab-

FIGURE 1. Lyn+/2 mice have a perturbed B cell

compartment and their B cells show impaired inhibi-

tory signaling. Representative flow cytometry of spleen

cells from young (10-wk-old) C57BL/6 (C57), Lyn+/2,

or Lyn2/2 mice. (A) Distribution of B220 and CD138

indicating B220lowCD138hi plasma cells (highlighted

with an ellipse) and B220 with IgM to determine B cell

numbers and amount of surface IgM (MFI indicated).

(B) Staining with Abs to CD21, CD23, and IgM to

distinguish follicular B cell and MZB cell subsets

(upper panels). CD23+ cells were gated to examine the

T2 cell population (lower panels). For data in (A) and

(B), absolute cell numbers of the indicated populations

were calculated from individual mice based on total

cell counts and flow cytometry and are shown beneath

the plots; horizontal bars denote the mean value of the

data points in the plot. (C) B220+ spleen cells were

gated to examine activation marker expression. MFI is

presented in graphical form as a ratio of the control.

Flow cytometry data are representative of three inde-

pendent experiments and a minimum of n = 4 mice.

(D) Proliferation of purified splenic B cells from con-

trol and Lyn+/2 mice in the presence of medium, 10

mg/ml F(ab9)2, 10 mg/ml anti-IgM, or 5 mg/ml LPS for

3 d. Data shown are representative of three indepen-

dent experiments. (E) Western blot analysis of purified

splenic B cells from control and Lyn+/2 mice stimu-

lated for 0, 5, or 30 min with 20 mg/ml anti-IgM and

probed with anti–p-Erk or anti-Erk as a loading con-

trol. Densitometry ratios of p-Erk/Erk are shown, and

the Western blot is representative of three independent

experiments *p , 0.05, **p , 0.01, ***p , 0.001

versus control with regard to both absolute cell number

and MFI.

1728 INHIBITORY SIGNALING MOLECULES IN AUTOIMMUNE DISEASE

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

errant T cell activation occurred in Lyn+/2 mice, in which changesin the B cell compartment are not as extreme as in Lyn2/2 mice.The splenic CD4 T cell compartment of young mice was assessedfor the activation markers CD62L and CD69, which are shed andupregulated, respectively, upon cellular activation (Fig. 2A). Wefound that CD62L levels on the cell surface were unchanged,whereas CD69 was slightly upregulated, indicating a minor in-crease in activation. However, assessment of aged Lyn+/2 micerevealed the T cell compartment to be significantly activatedcompared with younger Lyn+/2 mice and aged control mice (Fig.2B). These proportional changes were mirrored by increases in theabsolute number of activated CD4 T cells (Fig. 2B).We found that T cell activation in aged Lyn2/2 mice correlates

with expansion of the myeloid compartment (35, 38). Therefore,we assessed the myeloid compartment in Lyn+/2 mice to deter-mine whether there were changes present that may be occurringprior to marked T cell activation. We did not observe any sig-nificant expansion of macrophages and granulocytes in youngLyn+/2 mice (Fig. 2C); however, in aged mice, myeloid expansionwas evident, and differences from control mice were statisticallysignificant (Fig. 2D). Thus, although myeloid expansion is a majorand early feature of Lyn2/2 mice (36, 38, 39), it is not a hallmarkof young Lyn+/2 mice.

ANA development and kidney disease occur in Lyn+/2 mice,albeit with delayed onset and severity

Although several changes in Lyn+/2 mice resemble homozygousdeficiency of Lyn, the apparently healthy appearance of Lyn+/2

mice and their longer lifespan (data not shown) brought intoquestion whether they were susceptible to the development ofautoimmune disease or whether they were protected as a resultof a reduced inflammatory environment, as exemplified by Lyn2/2

IL-62/2 mice (38, 43). In previous studies, we observed thatBALB/c background Lyn+/2 mice develop ANAs with age, but wedid not assess disease development (35). To determine whetherC57BL/6 background Lyn+/2 mice were prone to autoimmune

disease, we examined ANA levels in serum at regular timeintervals (Fig. 3A). We found that Lyn+/2 mice accumulatedANAs in serum, albeit at a markedly slower rate compared withLyn2/2 mice [cf. Lyn2/2 maximal titers at 30 wk (38)]. We alsoassessed the extent to which aged Lyn+/2 mice develop glomer-ulonephritis and found that, at 1 y of age, glomeruli in the kidneysof Lyn+/2 mice showed mild signs of disease (Fig. 3B), althoughthe disease was not as severe as that in matched Lyn2/2 mice (Fig.3B). Quantification of glomerular size as a disease indicator (40)revealed that Lyn+/2 glomeruli were not significantly enlargedcompared with control mice at 40–50 wk; an age at which Lyn2/2

mice have extensive glomerulonephritis. However, significantpathology, as indicated by glomerular enlargement, was evidentin Lyn+/2 mice at .60 wk of age (Fig. 3C).

Genetic association of Lyn and SHP-1 in autoimmunity

Lyn was also shown to interact with SHP-1 in inhibitory signalingin B cells (16, 17), so we next examined whether there was a ge-netic interaction between Lyn and SHP-1 in autoimmunity. Micecarrying heterozygote loss-of-function mutations in Lyn andSHP-1 were generated previously, with B cells from Lyn+/2Mev+/2

mice showing downregulated expression of IgM; however, theeffect of this B cell phenotype on autoimmune disease sus-ceptibility or the effect of these mutations on other cellularcompartments was not assessed (17). Thus, we produced double-heterozygous Lyn+/2Mev+/2 mice and sampled their serum reg-ularly to test for the presence of IgG ANAs, comparing their levelswith those of control and single-heterozygous mice (Fig. 4A).Although previous studies showed that heterozygote Mev+/2 miceare healthy and show only very subtle immune system defects (44,45), we found that Mev+/2 mice developed ANAs in their serum ina manner similar to Lyn+/2 mice (Figs. 3A, 4A). Strikingly, whenLyn+/2 and Mev+/2 mutations were compounded, ANA titers weredrastically increased (Fig. 4A). In some mice, the titers werenear the maximal detectable limit at only 20 wk of age, a featurecharacteristic of Lyn2/2 mice, which develop severe disease (38).

FIGURE 2. Aged Lyn+/2 mice show expanded

representation of activated T cells and myeloid cells.

Representative flow cytometry of spleen cells from

young (10-wk-old) (A, C) and aged (30-wk-old)

(B, D) control or Lyn+/2 mice stained with the in-

dicated Abs. (A, B) CD4+ spleen cells were gated

and assessed for expression of activation markers

CD62L (upper panels) and CD69 (lower panels).

(C, D) Staining of spleen cells with Abs to myeloid

cell markers. For data in (B) and (D), absolute cell

numbers of the indicated populations were calcu-

lated from individual mice based on total cell counts

and flow cytometry; horizontal bars denote the mean

value of the data points in the plot. Data are rep-

resentative of three independent experiments and a

minimum of n = 8 mice. *p , 0.05, **p , 0.01,

***p , 0.001.

The Journal of Immunology 1729

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

To assess pathological signs of disease, we studied kidney sectionsof aged control, Lyn+/2, Mev+/2, and Lyn+/2Mev+/2 mice anddetermined glomerular cross-sectional area (Fig. 4B, 4C). Glo-meruli from 40–50-wk-old Lyn+/2 mice and Mev+/2 mice showedsome minor changes but were not significantly enlarged. In con-trast, glomeruli from matched Lyn+/2Mev+/2 mice were globularand significantly enlarged (Fig. 4B, 4C), indicating that whenheterozygous mutations in Lyn and SHP-1 were compounded, theeffect on kidney pathology was pronounced. In 60–90-wk-oldmice, the glomeruli of both Lyn+/2 and Mev+/2 mice were sig-nificantly enlarged compared with controls, whereas the glomeruliof Lyn+/2Mev+/2 mice showed further enlargement and remainedsignificantly larger than did those of Lyn+/2 mice (Fig. 4C).

Mice with heterozygous deficiencies in Lyn and SHP-1 showexaggerated B cell perturbations

Because haploinsufficiency of Lyn and SHP-1 strongly influencedpredisposition to autoimmune disease, we examined various im-mune cell compartments in Lyn+/2Mev+/2 mice to provide

a possible cellular basis for this mechanism. We first assessedB cell subpopulations in the spleen by flow cytometry (Fig. 5);although there was no difference in spleen weight among anygroups (data not shown), the numbers of splenic B cells in Lyn+/2

Mev+/2 mice were significantly reduced, although B cell numbersin single heterozygote animals were unchanged (Fig. 5A). Inter-estingly, the expansion of plasma cells observed in Lyn+/2 micewas not exaggerated in compound Lyn+/2Mev+/2 heterozygotemice, despite mice with a heterozygous loss-of-function mutationin SHP-1 (Mev+/2) showing a minor, yet significant, expansion ofthe plasma cell compartment (Fig. 5A). The MZB and T2 pop-ulations were significantly diminished in Lyn+/2Mev+/2 micecompared with control mice, approaching the low numbers ob-served in Lyn2/2 mice (compare Fig. 1B versus Fig. 5B). In-triguingly, Mev+/2 mice showed considerable variation in theirMZB and T2 B cell compartments, with some mice showingnormal numbers and others showing severe diminution, makingthe differences from control mice not significant (an extreme ex-ample is shown in Fig. 5B).To determine whether the exaggerated B cell population changes

occurring in compound Lyn+/2Mev+/2 heterozygote mice wereaccompanied by increased B cell activation, additional cell surfacemarkers were assessed by flow cytometry. These studies showedthat both Lyn+/2 and Mev+/2 B cells had reduced surface IgMexpression, as observed previously (17). Interestingly, the amountof IgM on Mev+/2 B cells was significantly lower than on Lyn+/2

B cells and almost identical to that on B cells from compoundLyn+/2Mev+/2 mice (Fig. 5A, 5C). To further assess the level ofB cell activation, we measured the expression of MHC class II andCD86, which were already increased on Lyn+/2 B cells (Fig. 1C).Levels were further increased on the surface of compound Lyn+/2

Mev+/2 heterozygote mice B cells, indicating significant activa-tion (Fig. 5C); however, there was no significant difference fromcontrol B cells in the expression of MHC class II and CD86 onMev+/2 B cells.

To determine whether the changes in the B cell compartment ofLyn+/2Mev+/2 mice had a biochemical basis, we stimulated pu-rified B cells from control, Lyn+/2, Mev+/2, and Lyn+/2Mev+/2

mice with intact IgM Ab, and phosphorylation of Erk was assessed(Fig. 5D). Enhanced and more sustained p-Erk was observed incompound Lyn+/2Mev+/2 B cells compared with Lyn+/2 B cells,which had enhanced and more sustained p-Erk compared withcontrol and Mev+/2 B cells. These data suggest that compoundmutant B cells showed enhanced signaling (Fig. 5D).

The T cell compartment is highly activated in aged Lyn+/2

Mev+/2 mice

We next examined the CD4 T cell compartment of young Lyn+/2

Mev+/2 mice, finding minimal differences (Fig. 6A). However, aspreviously observed in Lyn2/2 mice (35, 38) and Lyn+/2 mice(Fig. 2B), T cell activation became apparent in aged Lyn+/2Mev+/2

mice; interestingly, these T cells showed almost complete lossof CD62L expression, indicating substantial activation (Fig. 6B).The T cells of aged Mev+/2 mice showed some evidence of ac-tivation compared with those from control mice; however, thiswas minor compared with the T cell activation observed in agedLyn+/2 mice or Lyn+/2Mev+/2 mice (Fig. 6B).

Myeloid expansion occurs in aged Lyn+/2Mev+/2 mice

The compound heterozygous loss-of-function mutations in Lynand SHP-1 had far greater effects on B and T cell activation thandid either mutation alone. We previously showed that, in C57BL/6Lyn2/2 mice, expansion of the myeloid compartment precedesperturbations in the T cell compartment (38). However, in Lyn+/2

FIGURE 3. Lyn+/2 mice develop ANAs and glomerulonephritis. (A)

IgG ANAs in the serum of C57BL/6 background control, Lyn+/2, and

Lyn2/2 mice at the indicated ages by ELISA. Values refer to the absor-

bance reading obtained at 450 nm with correction at 595 nm; horizontal

bars denote the mean value of the data points at each time point. (B) H&E-

stained sections through the cortex of kidneys from 40–50-wk-old C57BL/

6, Lyn+/2, and Lyn2/2 mice and 80-wk-old C57BL/6 and Lyn+/2 mice.

Scale bars, 50 mm. Representative specimens were chosen for each ge-

notype. (C) Quantification of glomerular area from the indicated mice in

(B). For each genotype, one point represents the mean glomerular area of

a single mouse taken from an average of 20–30 glomeruli/mouse. Hori-

zontal bars represent the mean glomerular area for that genotype where n =

number of points on the graph (n $ 4). **p , 0.01, ***p , 0.001.

1730 INHIBITORY SIGNALING MOLECULES IN AUTOIMMUNE DISEASE

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

mice, myeloid expansion does not occur until the mice are aged(Fig. 2D). When we examined the myeloid compartment of youngLyn+/2Mev+/2 mice, no changes were apparent, unlike in youngLyn2/2 mice (Fig. 6C). However, in aged Lyn+/2Mev+/2 mice,there was a considerable increase in numbers of both granulocytes(Mac-1+Gr-1+) and macrophages (Mac-1+c-fms+) in Lyn+/2Mev+/2

mice. This expansion in the myeloid compartment was signifi-cantly exaggerated over that observed in aged Lyn+/2 mice (Fig.6D) and resembled that previously observed in age-matched 30-wk-old Lyn2/2 mice (38). Of note, there was no expansion in themyeloid compartment of aged Mev+/2 mice.

Minor myeloid progenitor expansion occurs in Lyn+/2Mev+/2

mice

Myeloid progenitor expansion is another component of auto-immune disease in Lyn2/2 mice (36, 38, 39). Young Lyn2/2

mice have augmented numbers of progenitors responsive tomyeloid cytokines, which increase dramatically as the mice ageand autoimmune disease becomes severe. Because Lyn+/2Mev+/2

mice only show myeloid cell expansion with age, we next de-termined whether myeloid progenitor cells were present at anincreased frequency in Lyn+/2Mev+/2 mice. In young Lyn+/2

Mev+/2 mice, splenic myeloid progenitors were increased, butthis increase was minor and not statistically significant com-pared with the large numbers of progenitors observed in thespleens of young Lyn2/2 mice (Fig. 7). Lyn+/2 and Mev+/2 micewere also included for comparison and showed no statisticallysignificant differences in myeloid progenitor numbers. Interest-ingly, aged Lyn+/2Mev+/2 mice showed a statistically significantincrease in splenic myeloid progenitors (Fig. 7), but this wasconsiderably less than the expansion observed in aged Lyn2/2

mice (36, 38, 39). Thus, although Lyn+/2Mev+/2 mice showmyeloid cell expansion similar to aged Lyn2/2 mice (Fig. 6D)(38), this occurs without the dramatic enhancement in myeloidprogenitor number.

Perturbations in the erythroid compartment occur in Lyn+/2

Mev+/2 mice

Finally, we analyzed the erythroid cell compartment in double-heterozygous Lyn+/2Mev+/2 mice, because our previous studiessuggested a close relationship between Lyn and SHP-1 in regu-lating erythropoiesis (36). Although we observed slight changes inthis compartment in young Lyn+/2, Mev+/2, and Lyn+/2Mev+/2

mice, the differences from control mice were not significant (Fig.8A). However, in aged mice, a significant expansion in Ter119+

CD71+ erythroblasts occurred in Lyn+/2Mev+/2 mice. Althoughthere was little change in aged Mev+/2 mice, aged Lyn+/2 miceshowed considerable variation in the numbers of erythroblasts,making the differences from control mice nonsignificant (Fig. 8B).

Genetic interaction between Lyn and SHIP-1 in autoimmunity

Previous studies showed that Lyn and the inositol phosphataseSHIP-1 are in the same biochemical pathway that negativelyregulates B cell signaling; membrane recruitment of SHIP-1 isseverely impaired in Lyn-deficient B cells through loss of Lyn-dependent FcgRIIB1 phosphorylation (18, 19). Because inhibi-tory signaling through FcgRIIB1 is also impaired in Lyn+/2

B cells (Fig. 1D, 1E), we next asked whether a genetic interactionexisted between Lyn and SHIP-1 in autoimmunity. For thesestudies, we generated double-heterozygote Lyn+/2SHIP-1+/2 miceon both the C57BL/6 and BALB/c genetic backgrounds. Whenmeasuring ANA titers, we had to use BALB/c genetic backgroundmice, because ANAs in C57BL/6 background SHIP-1 mutantmice are not detectable using a commercial ELISA kit (27). Wefound that, although BALB/c Lyn+/2 mice developed high ANAsin their serum, SHIP-1+/2 mice did not, even when close to a yearin age (Fig. 9A). Nonetheless, when the SHIP-1+/2 mutation wascompounded with the Lyn+/2 mutation, the double-heterozygotemice developed ANAs at a significantly faster rate, demonstratinga clear genetic interaction between Lyn and SHIP-1 in the de-velopment of autoimmunity. To assess whether the production of

FIGURE 4. Genetic interaction between Lyn and

SHP-1 in autoimmune disease development. (A) IgG

ANAs in serum of C57BL/6, Lyn+/2, Mev+/2, and

Lyn+/2Mev+/2 mice at the indicated ages. Values

refer to the absorbance reading obtained at 450 nm

with correction at 595 nm; horizontal bars denote

the mean value of the data points in each group.

(B) H&E-stained sections through the cortex of

kidneys of 48–52-wk-old C57BL/6, Lyn+/2, Mev+/2,

and Lyn+/2Mev+/2 mice. Shown are representative

examples of each genotype. Scale bar, 50 mm. (C)

Quantification of glomerular area from 40–50- and

60–90-wk-old C57BL/6, Lyn+/2, Mev+/2, and Lyn+/2

Mev+/2 mice. For each genotype, one point repre-

sents the mean glomerular area of a single mouse

taken from an average of 20–30 glomeruli. Hori-

zontal bars represent the mean glomerular area for

that genotype where n = number of points on the

graph (n $ 4). *p , 0.05, **p , 0.01, ***p ,0.001.

The Journal of Immunology 1731

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

ANAs correlated with SLE-like disease in the form of glomeru-lonephritis, kidney sections from age-matched BALB/c back-ground mice were examined. Surprisingly, despite developinghigh titers of ANAs, BALB/c background Lyn+/2 did not developsignificant glomerulonephritis, even when .1 y of age, withkidney disease confined to slight, but not significant, glomerularenlargement (Fig. 9B, 9D; 70-wk-old mice shown). However,BALB/c Lyn+/2SHIP-1+/2 mice displayed signs of mild glomer-ulonephritis, with glomeruli being significantly enlarged com-pared with controls and single-heterozygote mice (Fig. 9B, 9D).To determine whether this effect was replicated on the C57BL/6background, we also assessed the kidneys of aged C57BL/6 Lyn+/2,SHIP-1+/2, and Lyn+/2SHIP-1+/2 mice (Fig. 9C, 9D). Unlikeon the BALB/c background, we observed mild, yet significant,kidney pathology in aged C57BL/6 Lyn+/2 and SHIP-1+/2 mice,with glomerular size being significantly increased compared withcontrols. Enhanced kidney pathology was observed in Lyn+/2

SHIP-1+/2 mice, in which glomeruli showed extensive leukocyticinfiltrates, globularity, and enlargement (Fig. 9C). In several mice,extensive kidney damage had occurred, as reflected by severelydistended glomeruli or empty spaces in the cortex where glo-meruli had been destroyed (one example shown in Fig. 9C).When glomerular size was measured, glomeruli were significantlylarger than were those of C57BL/6 Lyn+/2 and SHIP-1+/2 mice(Fig. 9D).

DiscussionThe importance of Lyn tyrosine kinase as a regulator of autoim-mune disease was first revealed in analyses of Lyn-deficient micein the mid-1990s (8, 9), and it is becoming increasingly clear inmore recent human disease studies (14, 15). We have examinedthe consequences of haploinsufficiency of Lyn, alone or incombination with heterozygous loss-of-function mutations ininhibitory-signaling molecules known to be activated by Lyn,on the development of inflammation and autoimmune disease.We show that Lyn+/2 mice develop many features of their ho-mozygous counterparts, albeit in a milder form. These includealterations in B cell subsets and plasma cells; increased cellularactivation, as indicated by downregulation of cell-surface IgMand increased MHC class II and CD86 levels; and heightenedB cell responsiveness to external stimuli. Furthermore, Lyn+/2

mice display age-dependent dysregulation of both T cell andmyeloid compartments, features reminiscent of Lyn2/2 mice. Fi-nally, Lyn+/2 mice on a C57BL/6 background develop an auto-immune disease that is clinically similar to that of Lyn2/2 mice.Collectively, these results show that Lyn is a key inhibitor ofautoimmune disease development and one that is extremely sensitiveto gene dose, clearly demonstrating that Lyn is a haploinsufficientgene for autoimmune disease.We also show that partial inactivation of key inhibitory mole-

cules downstream of Lyn, specifically SHIP-1 and SHP-1, amplifiesthe consequences of Lyn insufficiency and leads to an acceleration

FIGURE 5. B cell population perturbations in Lyn+/2 mice are exacer-

bated in Lyn+/2Mev+/2 mice. Representative flow cytometry of spleen

cells from young (10-wk-old) control, Lyn+/2, Mev+/2, and Lyn+/2Mev+/2

mice. (A) Staining with Abs to B220 and CD138 to detect B220lowCD138hi

plasma cells (population highlighted with an ellipse) and Abs to B220 and

IgM to determine proportions of B cells and levels of surface IgM (MFI

indicated). (B) Staining with Abs to CD21, CD23, and IgM to distinguish

follicular zone B and MZB cell subsets (upper panels). Gates were set on

CD23+ cells to examine the T2 cell population (lower panels). For data in

(A) and (B), absolute cell numbers of the indicated populations were cal-

culated from individual mice based on total cell counts and flow cytom-

etry; horizontal bars denote the mean value of the data points in each group.

(C) B220+ spleen cells were gated to examine activation marker expres-

sion. MFI is presented in graphical form as a ratio of the control. For all

flow cytometry data, plots shown are representative of three independent

experiments and a minimum of n = 7 mice. (D) Western blot analysis of

purified splenic B cells from control, Lyn+/2, Mev+/2, and Lyn+/2Mev+/2

mice stimulated for 0, 5, or 30 min with 20 mg/ml anti-IgM and probed

with anti–p-Erk or anti-Erk as a loading control. Densitometry ratios of p-

Erk/Erk are shown, and the Western blot is representative of two inde-

pendent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

1732 INHIBITORY SIGNALING MOLECULES IN AUTOIMMUNE DISEASE

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

of disease development. However, within these pathways, Lynappears to be the definitive regulatory component, because thereduction in active Lyn is required for disease development,whereas haploinsufficiency of SHIP-1 or SHP-1 alone is of limitedconsequence. These studies clearly show that Lyn expression needsto be tightly regulated to achieve immune homeostasis.Recently, it was proposed that the myeloid compartment plays

a crucial role in the development of autoimmunity in Lyn2/2 mice(46). The investigators suggested that deregulated BAFF produc-tion by Lyn-deficient myeloid cells promoted T cell activation andthe induction of IFN-g secretion, which is then able to sustainmyeloid cell activation and BAFF production. This does not appearto be a complete explanation of disease development because theB cell compartment of Lyn2/2 mice does not resemble that ofBAFF transgenic mice, which show expanded follicular B2 andMZB cells (4), two populations that are absent in Lyn2/2 mice andcompromised in Lyn+/2 mice. Previous studies showed that Lyn2/2

mice on either mixed or C57BL/6 backgrounds show early-onsetmyeloid hyperplasia (36, 38, 39). These myeloid compartmentchanges correlate with early-onset ANAs in serum; however, at thisearly time point, their T cell compartment is ostensibly normal andonly becomes perturbed as the mice age and develop disease (38).Intriguingly, young C57BL/6 Lyn+/2 mice have normal numbers ofmyeloid cells and only develop an expanded myeloid compartmentas they age. These observations suggest that, in the Lyn+/2 setting,age-dependent perturbations in the T cell compartment occur in theabsence of early myeloid cell defects, implying that these effectsmay be driven by B cell defects initially, in addition to eventualperturbations in myeloid cells.

FIGURE 6. T cell and myeloid changes are exaggerated in aged Lyn+/2

Mev+/2 mice. Representative flow cytometry of spleen cells from young

(10-wk-old) (A, C) and aged (30-wk-old) (B, D) control, Lyn+/2, Mev+/2,

and Lyn+/2Mev+/2 mice. (A, B) CD4+ spleen cells were gated and assessed

for expression of CD62L. (C, D) Staining of spleen cells with Abs to

myeloid cell markers. For data in (C) and (D), absolute cell numbers of the

indicated populations were calculated based on total cell counts and flow

cytometry; horizontal bars denote the mean value of the data points in each

group. For all flow cytometry data, plots shown are representative of three

independent experiments and a minimum of n = 6 mice. *p , 0.05, **p ,0.01, ***p , 0.001.

FIGURE 7. Moderate myeloid progenitor expansion occurs in aged

Lyn+/2Mev+/2 mice. Quantitation of myeloid progenitors in the indicated

young (10-wk-old) (A) and aged (30-wk-old) (B) mice. In young mice,

Lyn2/2 mice were included for comparison. The data shown are repre-

sentative of n = 4 mice in two independent experiments and are presented

as mean 6 SEM. **p , 0.01 versus control.

The Journal of Immunology 1733

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

Interestingly, early expansion of plasma cells in Lyn+/2 mice didnot result in production of IgG ANAs in young mice, in contrast toLyn2/2 mice, a feature also observed by other investigators (43).It appears that plasma cells do not produce ANAs in the absenceof an inflammatory environment (early myeloid expansion).This is reminiscent of Lyn2/2IL-62/2 mice, in which significantnumbers of plasma cells are present; however, because of theabsence of IL-6–mediated inflammatory components, they lackIgG ANAs and remain disease-free throughout life (38, 43). Incontrast, in aged Lyn+/2 mice, myeloid expansion and significantT cell activation eventually come to the fore, concomitant withthe production of pathogenic IgG ANAs, leading to nephritis. Wepreviously found that Lyn+/2 mice on the BALB/c genetic back-ground develop IgG autoantibodies (35). In this study, we reca-pitulated these findings on the C57BL/6 background while alsodemonstrating that these mice develop autoimmune disease, albeitwith a longer latency and a reduced severity relative to Lyn2/2

mice. Another study failed to detect IgG autoantibodies in Lyn+/2

mice (43); however, they only examined mice up to 18 wk of age,a time point at which IgG autoantibodies in C57BL/6 backgroundLyn+/2 mice are low (Fig. 3A). This milder, yet significant, dis-ease phenotype observed in mice with a heterozygous mutationin Lyn is reminiscent of human diseases that also result fromheterozygous, rather than null, mutations in critical genes. Forinstance, heterozygous loss-of-function mutations in the sialicacid esterase gene have been identified in rheumatoid arthritis andtype 1 diabetes, indicating that these mutations act in a dominantfashion to influence disease (47). In addition, a heterozygousmutation in TNFRSF1A was associated with SLE in Japanesepatients (48), whereas heterozygous mutations in AIRE wereshown to be associated with multiple autoimmune syndromes,

including hepatitis (49) and hypoparathyroidism (50). To extrap-olate from our studies in mice, we suggest that there may bea subset of SLE patients that shows loss of one allele of Lyn,or at least single nucleotide polymorphisms in regulatory ele-ments that affect Lyn gene expression. Indeed, this is supported,in part, by studies that show reduced expression of Lyn in lupuspatients (11–13).Haploinsufficiency of both Lyn and SHP-1 was reported pre-

viously (17); however, the extent to which the mice developedautoimmunity and associated disease was not assessed. Weshowed a clear genetic interaction between Lyn and SHP-1 in thedevelopment of autoimmune disease. Lyn+/2Mev+/2 mice developautoantibodies and glomerulonephritis with age, showing signs ofdisease that are exaggerated over Lyn+/2 mice. The strength ofthis genetic interaction is also mirrored by IgG ANA titers, be-cause those of 18–22-wk-old Lyn+/2Mev+/2 mice resemble thoseof Lyn2/2 mice (38). In addition, T cell activation is a majorfeature of aged Lyn+/2Mev+/2 mice, resembling the T cell com-partment of aged Lyn2/2 mice. However, certain aspects of dis-ease in Lyn+/2Mev+/2 mice differ from those in Lyn2/2 mice.Myeloid cell expansion, which is observed in young Lyn2/2 mice,is only significant in aged Lyn+/2Mev+/2 mice; interestingly, thisis not reflected by a dramatic increase in splenic myeloid pro-genitors, a feature of Lyn2/2 mice (38). A possible explanation isthat expansion of the mature myeloid cell compartment and theexpansion of myeloid progenitor cells require distinct forms of aninflammatory environment, which may differ between Lyn2/2 andLyn+/2Mev+/2 mice. Assessment of indices of inflammation, in-cluding myeloid effector cell function and cytokine environment,may be useful in delineating the key differences between Lyn+/2

Mev+/2 and Lyn2/2 mice, as would a comparative analysis of dis-ease onset and degree in Lyn+/2Mev+/2 mice versus Lyn2/2 mice.SHIP-1 is a key regulatory molecule known to be downstream of

Lyn in inhibitory signaling (10, 18, 19). As a further example ofcombining heterozygous loss-of-function mutations on the Lyn-signaling pathway, we studied autoimmune disease developmentin Lyn+/2SHIP-1+/2 mice. ANA titers were increased consider-ably in BALB/c background Lyn+/2SHIP-1+/2 mice comparedwith single-heterozygote mice, with this interaction being mir-rored in the development of kidney pathology. Neither Lyn+/2 norSHIP-1+/2 BALB/c background mice developed significant glo-merular pathology, even when .1 year old, whereas BALB/cbackground Lyn+/2SHIP-1+/2 compound heterozygote mice dis-played mild, but significant, signs of glomerulonephritis, demon-strating a clear genetic interaction between Lyn and SHIP-1 inautoimmune disease development. The mild pathology is in starkcontrast to C57BL/6 background Lyn+/2SHIP-1+/2 mice, whichdeveloped severe kidney pathology consisting of extensive leu-kocytic infiltrates and glomerular destruction. Intriguingly, whencomparing kidney disease in Lyn+/2 mice on the two differentgenetic backgrounds, the extent of kidney pathology showed littlerelationship to ANA titers; BALB/c Lyn+/2 mice developed highANA titers without significant disease, whereas C57BL/6 back-ground Lyn+/2 mice developed low-moderate ANA titers butexhibited marked pathology. Therefore, ANA levels may providean indication of autoimmunity but are not a true measure ofautoimmune-mediated kidney pathology. The apparent resistanceof the BALB/c background to severe glomerulonephritis, despitethe presence of high-titer IgG ANAs, is supported by mild kidneydisease in BALB/c Lyn2/2 mice and BALB/c SHIP-12/2 mice,both of which develop high-titer IgG ANAs by 30–40 wk of age(27, 35). These studies confirm that the BALB/c genetic back-ground itself carries lupus-suppressor loci, as was suggested pre-viously (27, 51, 52).

FIGURE 8. Erythroid expansion occurs in aged Lyn+/2Mev+/2 mice.

Representative three-color flow cytometry of spleen cells from young (10-

wk-old) (A) and aged (30-wk-old) (B) control, Lyn+/2, Mev+/2, and Lyn+/2

Mev+/2 mice stained with the indicated Abs to erythroid cell markers. For

data in (B), absolute cell numbers of the indicated populations were cal-

culated based on total cell counts and flow cytometry and are shown below

the figure; horizontal bars denote the mean value of the data points in each

group. For all flow cytometry data, plots shown are representative of three

independent experiments and a minimum of n = 6 mice. ***p = 0.0004.

1734 INHIBITORY SIGNALING MOLECULES IN AUTOIMMUNE DISEASE

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

Collectively, these studies highlight the importance of the Lyntyrosine kinase as an autoimmune-susceptibility gene, showing thatreducing Lyn expression by 50% has profound effects on the B cellcompartment and is sufficient to drive myeloid expansion, T cellactivation, and, ultimately, autoimmune disease on a susceptiblegenetic background. Although autoimmune disease is slower todevelop under conditions of Lyn haploinsufficiency, multiple ge-netic mutations in Lyn-regulated pathways can accelerate andexacerbate autoantibody development and the clinical manifes-tations of autoimmune disease, although interactions with geneticbackground also contribute to disease susceptibility. Closer ex-amination of Lyn and Lyn-regulated pathways in human disease isclearly warranted.

AcknowledgmentsWe thank Val Feakes and Cary Tsui for histology preparations, Cathy Qui-

lici for technical assistance, and the staff from the animal facilities for

animal husbandry. We are grateful to Prof. Catriona McLean, Head of An-

atomical Pathology, Alfred Hospital (Melbourne, VIC, Australia), for ad-

vice about pathological assessment of renal disease and to the staff at the

Centre for Advanced Microscopy and Monash Micro Imaging facilities at

the Ludwig Institute for Cancer Research and Monash University, respec-

tively, for microscopy resources and assistance.

DisclosuresThe authors have no financial conflicts of interest.

References1. Fairhurst, A. M., A. E. Wandstrat, and E. K. Wakeland. 2006. Systemic lupus

erythematosus: multiple immunological phenotypes in a complex genetic dis-ease. Adv. Immunol. 92: 1–69.

2. Tedder, T. F., M. Inaoki, and S. Sato. 1997. The CD19-CD21 complex regulatessignal transduction thresholds governing humoral immunity and autoimmunity.Immunity 6: 107–118.

3. Sato, S., M. Fujimoto, M. Hasegawa, K. Takehara, and T. F. Tedder. 2004. Al-tered B lymphocyte function induces systemic autoimmunity in systemic scle-rosis. Mol. Immunol. 41: 1123–1133.

4. Mackay, F., S. A. Woodcock, P. Lawton, C. Ambrose, M. Baetscher,P. Schneider, J. Tschopp, and J. L. Browning. 1999. Mice transgenic for BAFFdevelop lymphocytic disorders along with autoimmune manifestations. J. Exp.Med. 190: 1697–1710.

5. Groom, J., S. L. Kalled, A. H. Cutler, C. Olson, S. A. Woodcock, P. Schneider,J. Tschopp, T. G. Cachero, M. Batten, J. Wheway, et al. 2002. Association of

FIGURE 9. Genetic interaction between Lyn and SHIP-1 in autoimmune disease development is influenced by genetic background. (A) IgG ANAs in the

serum of BALB/c background control, Lyn+/2, SHIP-1+/2, and Lyn+/2SHIP-1+/2 mice at the indicated ages were quantified by ELISA. Values refer to the

absorbance reading obtained at 450 nm with correction at 595 nm; horizontal bars denote the mean value of the data points in each group. H&E-stained

sections through the cortex of kidneys of 56–70-wk-old BALB/c background (B) and C57BL/6 background (C) control, Lyn+/2, SHIP-1+/2, and Lyn+/2

SHIP-1+/2 mice. Shown are representative examples of control, Lyn+/2, and SHIP-1+/2 mice and two examples of Lyn+/2SHIP-1+/2 mice. Scale bars, 50

mm. (D) Quantification of glomerular area from the indicated mice in (B) and (C) on BALB/c versus C57BL/6 genetic backgrounds. For each genotype, one

point represents the mean glomerular area of a single mouse taken from an average of 20–30 glomeruli. The horizontal bar represents the mean glomerular

area for that genotype, where n = number of points on the graph (n $ 4). *p , 0.05, **p , 0.01, ***p , 0.001.

The Journal of Immunology 1735

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from

BAFF/BLyS overexpression and altered B cell differentiation with Sjogren’ssyndrome. J. Clin. Invest. 109: 59–68.

6. Lewis, M. J., and M. Botto. 2006. Complement deficiencies in humans andanimals: links to autoimmunity. Autoimmunity 39: 367–378.

7. Xu, Y., K. W. Harder, N. D. Huntington, M. L. Hibbs, and D. M. Tarlinton. 2005.Lyn tyrosine kinase: accentuating the positive and the negative. Immunity 22: 9–18.

8. Hibbs, M. L., D. M. Tarlinton, J. Armes, D. Grail, G. Hodgson, R. Maglitto,S. A. Stacker, and A. R. Dunn. 1995. Multiple defects in the immune system ofLyn-deficient mice, culminating in autoimmune disease. Cell 83: 301–311.

9. Nishizumi, H., I. Taniuchi, Y. Yamanashi, D. Kitamura, D. Ilic, S. Mori,T. Watanabe, and T. Yamamoto. 1995. Impaired proliferation of peripheralB cells and indication of autoimmune disease in lyn-deficient mice. Immunity 3:549–560.

10. Hibbs, M. L., K. W. Harder, J. Armes, N. Kountouri, C. Quilici, F. Casagranda,A. R. Dunn, and D. M. Tarlinton. 2002. Sustained activation of Lyn tyrosinekinase in vivo leads to autoimmunity. J. Exp. Med. 196: 1593–1604.

11. Liossis, S. N., E. E. Solomou, M. A. Dimopoulos, P. Panayiotidis,M. M. Mavrikakis, and P. P. Sfikakis. 2001. B-cell kinase lyn deficiency inpatients with systemic lupus erythematosus. J. Investig. Med. 49: 157–165.

12. Huck, S., R. Le Corre, P. Youinou, and M. Zouali. 2001. Expression of B cellreceptor-associated signaling molecules in human lupus. Autoimmunity 33: 213–224.

13. Flores-Borja, F., P. S. Kabouridis, E. C. Jury, D. A. Isenberg, and R. A. Mageed.2005. Decreased Lyn expression and translocation to lipid raft signaling domainsin B lymphocytes from patients with systemic lupus erythematosus. ArthritisRheum. 52: 3955–3965.

14. Harley, J. B., M. E. Alarcon-Riquelme, L. A. Criswell, C. O. Jacob,R. P. Kimberly, K. L. Moser, B. P. Tsao, T. J. Vyse, C. D. Langefeld, S. K. Nath,et al; International Consortium for Systemic Lupus Erythematosus Genetics(SLEGEN). 2008. Genome-wide association scan in women with systemic lupuserythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 andother loci. Nat. Genet. 40: 204–210.

15. Lu, R., G. S. Vidal, J. A. Kelly, A. M. Delgado-Vega, X. K. Howard,S. R. Macwana, N. Dominguez, W. Klein, C. Burrell, I. T. Harley, et al; BIO-LUPUS and GENLES Multicenter Collaborations. 2009. Genetic associations ofLYN with systemic lupus erythematosus. Genes Immun. 10: 397–403.

16. Smith, K. G., D. M. Tarlinton, G. M. Doody, M. L. Hibbs, and D. T. Fearon.1998. Inhibition of the B cell by CD22: a requirement for Lyn. J. Exp. Med. 187:807–811.

17. Cornall, R. J., J. G. Cyster, M. L. Hibbs, A. R. Dunn, K. L. Otipoby, E. A. Clark,and C. C. Goodnow. 1998. Polygenic autoimmune traits: Lyn, CD22, and SHP-1are limiting elements of a biochemical pathway regulating BCR signaling andselection. Immunity 8: 497–508.

18. Chan, V. W., C. A. Lowell, and A. L. DeFranco. 1998. Defective negative reg-ulation of antigen receptor signaling in Lyn-deficient B lymphocytes. Curr. Biol.8: 545–553.

19. Nishizumi, H., K. Horikawa, I. Mlinaric-Rascan, and T. Yamamoto. 1998. Adouble-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals. J. Exp. Med. 187: 1343–1348.

20. Ho, L. H., T. Uehara, C. C. Chen, H. Kubagawa, and M. D. Cooper. 1999.Constitutive tyrosine phosphorylation of the inhibitory paired Ig-like receptorPIR-B. Proc. Natl. Acad. Sci. USA 96: 15086–15090.

21. Maeda, A., A. M. Scharenberg, S. Tsukada, J. B. Bolen, J. P. Kinet, andT. Kurosaki. 1999. Paired immunoglobulin-like receptor B (PIR-B) inhibitsBCR-induced activation of Syk and Btk by SHP-1. Oncogene 18: 2291–2297.

22. Helgason, C. D., J. E. Damen, P. Rosten, R. Grewal, P. Sorensen, S. M. Chappel,A. Borowski, F. Jirik, G. Krystal, and R. K. Humphries. 1998. Targeted dis-ruption of SHIP leads to hemopoietic perturbations, lung pathology, anda shortened life span. Genes Dev. 12: 1610–1620.

23. Liu, Q., T. Sasaki, I. Kozieradzki, A. Wakeham, A. Itie, D. J. Dumont, andJ. M. Penninger. 1999. SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev. 13: 786–791.

24. Liu, Q., A. J. Oliveira-Dos-Santos, S. Mariathasan, D. Bouchard, J. Jones,R. Sarao, I. Kozieradzki, P. S. Ohashi, J. M. Penninger, and D. J. Dumont. 1998.The inositol polyphosphate 5-phosphatase ship is a crucial negative regulator ofB cell antigen receptor signaling. J. Exp. Med. 188: 1333–1342.

25. Brauweiler, A., I. Tamir, J. Dal Porto, R. J. Benschop, C. D. Helgason,R. K. Humphries, J. H. Freed, and J. C. Cambier. 2000. Differential regulation ofB cell development, activation, and death by the src homology 2 domain-containing 59 inositol phosphatase (SHIP). J. Exp. Med. 191: 1545–1554.

26. Helgason, C. D., C. P. Kalberer, J. E. Damen, S. M. Chappel, N. Pineault,G. Krystal, and R. K. Humphries. 2000. A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant developmentand enhanced function of b lymphocytes in ship 2/2 mice. J. Exp. Med. 191:781–794.

27. Maxwell, M. J., M. Duan, J. E. Armes, G. P. Anderson, D. M. Tarlinton, andM. L. Hibbs. 2011. Genetic segregation of inflammatory lung disease and au-toimmune disease severity in SHIP-12/2 mice. J. Immunol. 186: 7164–7175.

28. Shultz, L. D., and M. C. Green. 1976. Motheaten, an immunodeficient mutant ofthe mouse. II. Depressed immune competence and elevated serum immunoglo-bulins. J. Immunol. 116: 936–943.

29. Shultz, L. D., D. R. Coman, C. L. Bailey, W. G. Beamer, and C. L. Sidman. 1984.“Viable motheaten,” a new allele at the motheaten locus. I. Pathology. Am. J.Pathol. 116: 179–192.

30. Croker, B. A., B. R. Lawson, S. Rutschmann, M. Berger, C. Eidenschenk,A. L. Blasius, E. M. Moresco, S. Sovath, L. Cengia, L. D. Shultz, et al. 2008.Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1,MyD88, and a microbial trigger. Proc. Natl. Acad. Sci. USA 105: 15028–15033.

31. O’Keefe, T. L., G. T. Williams, S. L. Davies, and M. S. Neuberger. 1996. Hy-perresponsive B cells in CD22-deficient mice. Science 274: 798–801.

32. Nitschke, L., R. Carsetti, B. Ocker, G. Kohler, and M. C. Lamers. 1997. CD22 isa negative regulator of B-cell receptor signalling. Curr. Biol. 7: 133–143.

33. O’Keefe, T. L., G. T. Williams, F. D. Batista, and M. S. Neuberger. 1999. De-ficiency in CD22, a B cell-specific inhibitory receptor, is sufficient to predisposeto development of high affinity autoantibodies. J. Exp. Med. 189: 1307–1313.

34. Walker, J. A., and K. G. Smith. 2008. CD22: an inhibitory enigma. Immunology123: 314–325.

35. Tsantikos, E., C. Quilici, K. W. Harder, B. Wang, H. J. Zhu, G. P. Anderson,D. M. Tarlinton, and M. L. Hibbs. 2009. Perturbation of the CD4 T cell com-partment and expansion of regulatory T cells in autoimmune-prone Lyn-deficientmice. J. Immunol. 183: 2484–2494.

36. Harder, K. W., C. Quilici, E. Naik, M. Inglese, N. Kountouri, A. Turner,K. Zlatic, D. M. Tarlinton, and M. L. Hibbs. 2004. Perturbed myelo/erythropoiesis in Lyn-deficient mice is similar to that in mice lacking the in-hibitory phosphatases SHP-1 and SHIP-1. Blood 104: 3901–3910.

37. Beavitt, S. J., K. W. Harder, J. M. Kemp, J. Jones, C. Quilici, F. Casagranda,E. Lam, D. Turner, S. Brennan, P. D. Sly, et al. 2005. Lyn-deficient mice developsevere, persistent asthma: Lyn is a critical negative regulator of Th2 immunity. J.Immunol. 175: 1867–1875.

38. Tsantikos, E., S. A. Oracki, C. Quilici, G. P. Anderson, D. M. Tarlinton, andM. L. Hibbs. 2010. Autoimmune disease in Lyn-deficient mice is dependent onan inflammatory environment established by IL-6. J. Immunol. 184: 1348–1360.

39. Harder, K. W., L. M. Parsons, J. Armes, N. Evans, N. Kountouri, R. Clark,C. Quilici, D. Grail, G. S. Hodgson, A. R. Dunn, and M. L. Hibbs. 2001. Gain-and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn inthe myeloid lineage. Immunity 15: 603–615.

40. Koh, A. E., S. W. Njoroge, M. Feliu, A. Cook, M. K. Selig, Y. E. Latchman,A. H. Sharpe, R. B. Colvin, and E. Paul. 2011. The SLAM family member CD48(Slamf2) protects lupus-prone mice from autoimmune nephritis. J. Autoimmun.37: 48–57.

41. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, and C. A. Lowell. 1997.Characterization of the B lymphocyte populations in Lyn-deficient mice and therole of Lyn in signal initiation and down-regulation. Immunity 7: 69–81.

42. Meade, J., C. Fernandez, and M. Turner. 2002. The tyrosine kinase Lyn is re-quired for B cell development beyond the T1 stage in the spleen: rescue by over-expression of Bcl-2. Eur. J. Immunol. 32: 1029–1034.

43. Gutierrez, T., K. E. Halcomb, A. J. Coughran, Q. Z. Li, and A. B. Satterthwaite.2010. Separate checkpoints regulate splenic plasma cell accumulation and IgGautoantibody production in Lyn-deficient mice. Eur. J. Immunol. 40: 1897–1905.

44. Kuntz, L., P. Fonteneau, and F. Loor. 1990. Partial expression of the viablemotheaten (mev) mutation in the heterozygous state: abnormal levels of serumimmunoglobulins and natural anti-ssDNA antibodies. J. Autoimmun. 3: 329–335.

45. Kamata, T., M. Yamashita, M. Kimura, K. Murata, M. Inami, C. Shimizu,K. Sugaya, C. R. Wang, M. Taniguchi, and T. Nakayama. 2003. src homology 2domain-containing tyrosine phosphatase SHP-1 controls the development ofallergic airway inflammation. J. Clin. Invest. 111: 109–119.

46. Scapini, P., Y. Hu, C. L. Chu, T. S. Migone, A. L. Defranco, M. A. Cassatella,and C. A. Lowell. 2010. Myeloid cells, BAFF, and IFN-gamma establish aninflammatory loop that exacerbates autoimmunity in Lyn-deficient mice. J. Exp.Med. 207: 1757–1773.

47. Surolia, I., S. P. Pirnie, V. Chellappa, K. N. Taylor, A. Cariappa, J. Moya, H. Liu,D. W. Bell, D. R. Driscoll, S. Diederichs, et al. 2010. Functionally defective germlinevariants of sialic acid acetylesterase in autoimmunity. Nature 466: 243–247.

48. Ida, H., E. Kawasaki, T. Miyashita, F. Tanaka, M. Kamachi, Y. Izumi, M. Huang,M. Tamai, T. Origuchi, A. Kawakami, et al. 2004. A novel mutation (T61I) in thegene encoding tumour necrosis factor receptor superfamily 1A (TNFRSF1A) ina Japanese patient with tumour necrosis factor receptor-associated periodicsyndrome (TRAPS) associated with systemic lupus erythematosus. Rheumatol-ogy (Oxford) 43: 1292–1299.

49. Lankisch, T. O., O. Mourier, E. M. Sokal, D. Habes, F. Lacaille, L. Bridoux-Henno, B. Hermeziu, C. Lenaerts, C. P. Strassburg, and E. Jacquemin. 2009.AIRE gene analysis in children with autoimmune hepatitis type I or II. J. Pediatr.Gastroenterol. Nutr. 48: 498–500.

50. Cervato, S., L. Morlin, M. P. Albergoni, S. Masiero, N. Greggio, C. Meossi,S. Chen, M. del Pilar Larosa, J. Furmaniak, B. Rees Smith, et al. 2010. AIREgene mutations and autoantibodies to interferon omega in patients with chronichypoparathyroidism without APECED. Clin. Endocrinol. (Oxf.) 73: 630–636.

51. Tarasenko, T., H. K. Kole, and S. Bolland. 2008. A lupus-suppressor BALB/clocus restricts IgG2 autoantibodies without altering intrinsic B cell-tolerancemechanisms. J. Immunol. 180: 3807–3814.

52. Keszei, M., Y. E. Latchman, V. K. Vanguri, D. R. Brown, C. Detre, M. Morra,C. V. Arancibia-Carcamo, E. Paul, S. Calpe, W. Castro, et al. 2011. Auto-antibody production and glomerulonephritis in congenic Slamf12/2 andSlamf22/2 [B6.129] but not in Slamf12/2 and Slamf22/2 [BALB/c.129] mice.Int. Immunol. 23: 149–158.

1736 INHIBITORY SIGNALING MOLECULES IN AUTOIMMUNE DISEASE

by guest on July 25, 2016http://w

ww

.jimm

unol.org/D

ownloaded from