Preferential production of IgG1, IL-4 andIL-10 in MuSK-immunized mice
Canan Ulusoya,1, Eunmi Kimb,1, Erdem Tüzüna,⁎, Ruksana Hudab,Vuslat Yılmazc, Konstantinos Poulasd, Nikos Trakase, Lamprini Skriapad,e,Athanasios Niarchosd, Richard T. Strait f, Fred D. Finkelmang,h,i,Selin Turana, Paraskevi Zisimopouloue, Socrates Tzartos d,e,Güher Saruhan-Direskeneli c, Premkumar Christadoss b
a Department of Neuroscience, Institute for Experimental Medical Research, University of Istanbul, Istanbul 34393, Turkeyb Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USAc Department of Physiology, Istanbul Faculty of Medicine, University of Istanbul, Istanbul 34393, Turkeyd Department of Pharmacy, School of Health Sciences, University of Patras, Patras 26504, Greecee Hellenic Pasteur Institute, Athens 115 21, Greecef Department of Pediatrics and Cincinnati Children's Hospital Division of Emergency Medicine, College of Medicine,University of Cincinnati, Cincinnati, OH 45229, USAg Department of Medicine, Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 45229, USAh Department of Internal Medicine, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USAi Division of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
Received 2 December 2013; accepted with revision 20 February 2014Available online 28 February 2014
KEYWORDSMyasthenia gravis;Experimentalautoimmune myastheniagravis;Muscle specific kinase;Anti-MuSK IgG1;IL-4;IL-10
Abstract Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weaknessassociated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predomi-nantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmunemyasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) andcytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30 μg ofrecombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significantEAMG susceptibility (N80% incidence). Althoughmice immunizedwith 10 μg of MuSK had lower EAMGincidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30 μgMuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement
fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions.Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Seraand MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly
of Neuroscience Institute for Experimental Medical Research University of Istanbul Vakıf Gureba Caddesi,4171.m (E. Tüzün).ually contributing joint first authors.
Myasthenia gravis (MG) is a well-characterized antibody-mediated autoimmune disease of the neuromuscular junction(NMJ). More than 80% of MG patients display serum antibodiesto acetylcholine receptor (AChR), which are predominantly ofthe immunoglobulin (Ig) G1 and IgG3 isotypes. AChR antibodiescause muscle weakness mainly by activation of the comple-ment cascade, subsequent destruction of the postsynaptic NMJand reduction of AChR expression [1,2]. Antibodies directedagainst muscle specific kinase (MuSK) are detected in around0–60% of AChR antibody negative MG patients [3]. In animals,passive transfer of MuSK antibodies and immunization withMuSK induce experimental autoimmune myasthenia gravis(EAMG) closely mimicking clinical and electrophysiologicalfeatures of MG, indicating that MuSK antibodies have a patho-genic action [4,5]. In contrast to AChR antibodies, MuSK anti-bodies are mainly of the non-complement fixing IgG4 isotype[6,7]. Therefore, MuSK antibodies disrupt NMJ functions notthrough complement mediated tissue destruction, but byinhibiting MuSK functions, consequently leading to impairedpostsynaptic AChR organization and reduced AChR expressionat the NMJ [8]. Although complement activating anti-MuSKIgG1 antibodies can be detected in the majority of MuSKantibody positive patients at low levels [7], passive transfer ofthese antibodies into experimental animals does not induceEAMG [4]. The absence of complement deposits at the NMJs ofMuSK antibody positive MG patients [9] further supports thenotion that in MuSK associated MG, muscle weakness is gen-erated by non-complement fixing anti-MuSK IgG4 antibodies.
Mouse models of MG have been extensively used for theidentification of pathogenic mechanisms of MG as well as theinvention of novel treatment methods for antibody mediateddisorders. As a result of these efforts, several cytokines (mostlyTh1-type and/or proinflammatory) have been found to be in-volved in AChR antibody production. Inborn or acquired defi-ciency of these cytokines in experimental animals often leadsto significant reduction in AChR antibody production and EAMGsusceptibility. Moreover, treatment trials targeting some ofthese cytokines have given promising results [10]. By contrast,very little is known about the immunological factors underlyingthe generation of MuSK antibodies. To uncover major immu-nological factors involved in MuSK immunity and by this wayidentify potential targets for future treatment trials, we ex-amined antibody and cytokine responses in MuSK immunizedmice. Our results suggest that MuSK, as an immunogen, pref-erentially promotes the production of IL-4 and IL-10 in mice,resulting in the production of IgG1, the mouse analog of humanIgG4 [11–13]. These findings might provide potential clues fornovel target-specific treatment methods (e.g. reagents spe-cifically inhibiting IL-4, IL-10 or IgG4) that do not cause globalimmunosuppression.
2. Materials and methods
2.1. Mice, MuSK, induction and clinical evaluation ofEAMG
Seven- to eight-week-old wild-type (WT) B6 mice were pur-chased from Jackson Laboratories (Bar Harbor, Maine, USA).Seven- to eight-week-old IgG1 KO mice in the C57BL/6 (B6)background were generated by targeted deletion of themurine IgG1 gene [14]. IgG1 KO mice were produced bybackcrossing to B6 mice for ten generations. The mice weredetermined to be completely IgG1 deficient by PCR, northernblot, immunohistochemistry analysis and serum IgG1 mea-surements. All animals were housed in the viral antibody-freebarrier facility at the University of Texas Medical Branch orIstanbul University and maintained according to the Institu-tional Animal Care and Use Committee Guidelines.
The extracellular domain (ECD) of humanMuSK (amino acids1–463, MUSK_HUMAN O15146-3) was cloned into the pPICZαAvector (Invitrogen, San Diego, CA) and was expressed in Pichiapastoris host strain X33 as soluble protein in the yeast culturesupernatant as described previously [15]. The expressedprotein was purified by metal affinity chromatography usingNi-NTA agarose resin (Qiagen, Valencia, CA), according to themanufacturer's protocol. Protein elution was performed usingan elution buffer containing 50 mM HEPES, 300 mM NaCl, 0.1%glycerol (pH 8.0) and increasing imidazole concentrations(20 mM, 50 mM, 100 mM, 150 mM, and 1 M). The recombinantMuSK-ECD eluted in the 150 mM imidazole fraction and furtherpurified on an AKTA 90 FPLC purifier system (Amersham-Pharmacia, Uppsala, Sweden), using a 24 ml Superose 12column (Amersham-Pharmacia). Protein concentration wasdetermined using the Bradford assay (Bio-Rad, Hercules, CA)[15,16]. The purity of the protein was documented by gelelectrophoresis and western blotting with a commercialanti-human MuSK antibody (Abcam, Cambridge, UK).
Mice were anesthetized and immunized with 10 or 30 μgof MuSK emulsified in complete Freund's adjuvant (CFA,Difco, Detroit, MI) s.c. at four sites (two hind footpads andshoulders) on day 0 and were boosted with the same amountof MuSK used in the first immunization in CFA or incompleteFreund's adjuvant (IFA, Difco) s.c. at four sites on the backon days 28 and 56. Control mice were immunized with onlyCFA. Mice were terminated 21 or 28 days after the 3rd im-munization depending on the severity of EAMG. For clinicalexamination, mice were left for 3 min on a flat platform andwere observed for signs of EAMG. Clinical muscle weaknesswas graded as follows: grade 0, mouse with normal posture,muscle strength, and mobility; grade 1, normal at rest, withmuscle weakness characteristically shown by a hunchedposture, restricted mobility, and difficulty raising the headafter exercise that consisted of 30 paw grips on a cage top
157Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice
grid; grade 2, grade 1 symptoms without exercise during theobservation period on a flat platform; grade 3, dehydratedand moribund with grade 2 weakness; and grade 4, dead. Forobjective measurement of muscle strength, mice were firstexercised with 40 paw grips on a cage top grid. Followingexercise, mice were made to grasp a grid attached to a dyna-mometer (Chatillon Digital Force Gauge, DFIS 2, ColumbusInstruments, Columbus, OH). The maximal force applied tothe dynamometer while pulling the mouse by its tail until itlost its grip on the grid was recorded.
2.2. ELISA for anti-MuSK Ig isotypes
Mice were bled from the tail vein 14 days after the last immu-nization. Sera were evaluated for anti-MuSK IgG, IgG1, IgG2b,IgG2c, IgG3 and IgM levels. Affinity-purified human MuSK(1 μg/ml) was coated onto 96-well microtiter plates in 0.1 Mcarbonate bicarbonate buffer overnight at 4 °C. Dilutedserum samples of 100 μl (1:1000) were added and incubatedat 37 °C for 90 min. Horseradish peroxidase (HRP)-conjugatedanti-mouse IgG, IgG1, IgG2b, IgG2c, IgG3 and IgM (Abcam)(1:10,000) were added and then incubated at 37 °C for90 min. Subsequently, the peroxidase indicator substrate 2,2′-azinobis-(3-ethylbenzothiazoline 6-sulfonate) substrate (ABTS)solution in 0.1 M citric buffer (pH 4.35) was added in thepresence of H2O2, and themixture was allowed to develop colorat room temperature in the dark. Plates were read at a wave-length of 405 nm. Since B6 mice do not express IgG2a [11], theanalogous isotype IgG2c was evaluated.
2.3. Immunofluorescence for Ig isotype, C3 and MACdeposits at NMJ
Sections (10 μm thick) were obtained from forelimb musclesamples of mice, frozen in liquid nitrogen, and stored at−80 °C. Slides were fixed in cold acetone and blocked in 10%normal goat serum in PBS. After washing with PBS, the sec-tions were incubated with tetramethylrhodamine-conjugatedbungarotoxin (BTx) (Molecular Probes, Eugene, OR) (1/500dilution) for 1 h at room temperature to label the NMJ.Sections were then incubated for 1 h at room temperaturewith FITC-conjugated antibodies to mouse IgG, IgG1, IgG2b,IgG2c, IgG3, IgM, complement C3 or C5b-9 (MAC) (Abcam)(diluted 1/1000) to colocalize Ig or complement deposits inNMJ. The sections were washed and viewed in a fluorescencemicroscope (Olympus IX-70). The number of Ig isotype-, C3-and MAC-positive BTx binding sites was counted in five musclesections from each mouse. The percentages of NMJs withdeposits in each muscle section were calculated by totalingthe numbers of deposits divided by the numbers of BTx labeledsites, times 100.
2.4. Cytokine measurements in sera and culturesupernatants
Inguinal, popliteal, and axillary lymph node cells (LNCs)were collected at the termination of the experiment. Cells(2 × 105 cells/well) were seeded in triplicate into 96-well,round-bottomed microtiter plates in 0.2 ml of RPMI 1640medium with MuSK (5 μg/ml) supplemented with 10% fetalcalf serum, penicillin G (100 U/ml) streptomycin (100 μg/ml),
L-glutamine (2 mM), 2-mercaptoethanol (3 × 10−5 M), andHEPES buffer (25 mM). As a control, LNCs from each mousewere incubated with culture medium only with no stimulatingprotein. The cells were cultured for 48 or 72 h at 37 °C inhumidified 5% CO2-enriched air. Supernatants were collectedand stored at −80 °C until analyzed. Levels of IL-4, IL-10,IL-12, IFN-γ, IL-6 and TNF-α in the supernatants were mea-sured by a Luminex Multiplex kit (Millipore, Billerica, MA),according to themanufacturer's instructions. For confirmationof this assay, serum levels of IL-4, IL-10, IL-12 and IFN-γ weremeasured by an ELISA kit (Peprotech, Rocky Hill, NJ) as per themanufacturer's instructions.
2.5. Statistical analysis
Clinical EAMG incidences were compared using the Fisher'sexact test. Clinical grades were compared by Mann–WhitneyU or Kruskal–Wallis tests (and Dunn's post-hoc test) for two-group and multiple-group comparisons, respectively. All otherparameters were compared using Student's t-test or ANOVA(and Tukey's post-hoc test) for two-group and multiple-groupcomparisons, respectively. p values less than 0.05 were con-sidered statistically significant.
3. Results
3.1. MuSK immunization predominantly inducesIgG1 production
To optimize the EAMG model induced by MuSK immunizationand to investigate Ig isotypes involved in MuSK immunity, WTB6 mice were immunized with 10 or 30 μg of MuSK in CFAor IFA. Clinical features and antibody responses of B6 miceimmunized with 30 μg MuSK in CFA in three successiveimmunizations (n = 6), with 30 μg MuSK in CFA in the firstimmunization and in IFA in the latter two immunizations(n = 7), with 10 μg MuSK in CFA in three successive immu-nizations (n = 7) and with only CFA in three successiveimmunizations (n = 6) were compared. Two-group compar-isons for clinical incidences were made by Fisher's exacttest. EAMG grades and grip strengths were respectivelycompared by Kruskal–Wallis with Dunn's post-hoc test andANOVA with Tukey's post-hoc test among four immunizationgroups. Starting from the 8th week after the first immuni-zation, mice immunized with 30 μg of MuSK in CFA or IFAshowed significantly higher EAMG incidence, clinical gradeand reduced grip strength than CFA immunized mice, where-as clinical parameters of mice immunized with 10 μg of MuSKin CFA were comparable to those of mice immunized withonly CFA throughout the study period (Figs. 1A–C). At termi-nation, 5 of 6 (83.3%) mice immunized with 30 μg MuSK inCFA in all immunizations and 6 of 7 (85.7%) mice immunizedwith 30 μg MuSK in CFA or IFA in different immunizations haddeveloped clinical EAMG, whereas only 1 of 7 (14.3%) miceimmunized with 10 μg MuSK in CFA in all immunizations andnone of the CFA immunized mice had developed noticeablemuscle weakness (Fig. 1A). There were no significant differ-ences in EAMG incidence (by Fisher's exact test), clinicalgrade (by Dunn's post-hoc test) and grip strength (by Tukey'spost-hoc test) of mouse groups immunized with 30 μg MuSKin CFA or CFA followed by IFA. Also, there were no significant
Figure 1 MuSK immunization induced EAMG and promoted preferential production of anti-MuSK IgG1 in B6 mice. Mice receiving30 μg MuSK in CFA in three successive immunizations [MuSK (30 μg) + CFA–CFA] and 30 μg MuSK in CFA in the first immunization andin IFA in the latter two immunizations [MuSK (30 μg) + CFA–IFA] showed comparable EAMG incidence (A), average clinical grade (B)and average grip strength (C) values, whereas mice receiving 10 μg of MuSK in CFA in three successive immunizations [MuSK(10 μg) + CFA–CFA] and mice immunized with CFA only in all immunizations were significantly resistant to EAMG induction. All mousegroups immunized with MuSK had significantly higher serum anti-MuSK IgG, IgG1 and IgG2b levels than mice immunized with CFA only(D). *, p b 0.05; **, p b 0.01; ***, p b 0.001 by Fisher's exact test, Kruskal–Wallis (and Dunn's post-hoc test), ANOVA (and Tukey'spost-hoc test) as required; vertical bars indicate standard errors. One representation of two independent experiments.
158 C. Ulusoy et al.
differences between mice immunized with 10 μg MuSK inCFA or with only CFA. These results indicated a dose depen-dent impact on clinical parameters induced by the MuSKimmunogen. Although exhibiting reduced clinical responses,serum anti-MuSK IgG, IgG1, IgG2b and IgM levels of miceimmunized with 10 μg MuSK in CFA were comparable tothose of mice immunized with 30 μg MuSK in CFA or IFA.Also, serum anti-MuSK IgG1 levels were relatively higher thananti-MuSK IgG2b and IgM levels in all MuSK immunized mousegroups (Fig. 1D).
3.2. IgG1 KO mice display reduced anti-MuSKIg responses
To further delineate the significance of the predominantIgG1 response in MuSK immunity, WT (n = 10) and IgG1 KO(n = 10) mice were immunized three times with 10 μg ofMuSK in CFA or only CFA (n = 10 for both WT and IgG1 KOmice) and serum samples were collected 14 days after thelast immunization. Similar to the first experiment, immuni-zation with 10 μg of MuSK induced significant anti-MuSKantibody responses and a predominant IgG1 productionin WT mice (Fig. 2) without generating appreciable clinical
muscle weakness (data not shown). IgG1 KO mice hadsignificantly lower anti-MuSK IgG and IgG2b responses thanWT mice, whereas serum anti-MuSK IgM levels of MuSKimmunized WT and IgG1 KO mice were identical. IgG1 KOmice did not exhibit anti-MuSK IgG1 production, as expected,confirming the IgG1 deficiency in this mouse strain. Miceimmunized with only CFA did not have anti-MuSK antibodies intheir sera (Fig. 2).
3.3. MuSK immunized mice exhibit complementfixing and non-complement fixing Ig isotypes in seraand NMJs
After optimizing the MuSK induced EAMG model in the firsttwo preliminary experiments, to have a complete image ofthe anti-MuSK isotype status, MuSK immunized mice wereinvestigated for the presence of a broader panel of antibodies inboth sera and NMJs. For this purpose, WT B6 mice were im-munized with MuSK in CFA (n = 8) or only CFA (n = 8) and theirserum and muscle samples were screened for several com-plement fixing (IgG2b, IgG2c, IgG3, IgM) and non-complementfixing (IgG1) isotypes. Mice immunized with 30 μg MuSK in CFAin three successive immunizations showed gradually increasing
Figure 2 Immunization with 10 μg of MuSK in CFA (MuSK-CFA) induced significantly reduced serum anti-MuSK IgG and IgG2bresponses in IgG1 knockout (KO) mice as compared to wild-type (WT) mice, whereas KO and WT mice immunized with CFA only (CFA)did not exhibit appreciable anti-MuSK antibody production, as expected. *, p b 0.05; **, p b 0.01; ***, p b 0.001 by ANOVA and Tukey'spost-hoc test; vertical bars indicate standard errors. One representation of two independent experiments.
159Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice
EAMG incidence, clinical grade and grip strength values asopposed to CFA immunized mice, reaching at termination to anoverall incidence of 87.5% (7 of 8 mice). Starting from the 5thweek to termination, EAMG incidences, clinical grades andgrip strengths of MuSK-CFA immunized mice were significantlyhigher than those of CFA immunizedmice as assessed by Fisher'sexact test, Mann–Whitney U test and Student's t-test, re-spectively. Notably, in this experiment two MuSK-CFA immu-nized mice started developing muscle weakness 3–4 weeksafter the first immunization without requiring the secondimmunization (Fig. 3A–C). MuSK-CFA immunized mice alsoshowed significant weight loss in the last two weeks of theexperiment as compared to CFA immunized mice (Fig. 3D).MuSK-CFA immunizedmice exhibited all investigated anti-MuSKIg isotypes in their sera, whereas CFA immunized mice did notshow anti-MuSK antibodies. Similar to the previous experi-ments, serum levels of anti-MuSK IgG1 were higher than thelevels of other isotypes (Fig. 3E). MuSK-CFA immunized butnot CFA immunized mice displayed NMJ deposits of both non-complement fixing IgG1 and complement fixing IgG2b, IgG2c,IgG3 and IgM. Similar to serum data, IgG1 deposits were moreprevalent at the NMJs than other isotypes (Figs. 3F, 4). Notably,C3 and MAC deposits were also detected at the NMJs ofMuSK-CFA immunized but not CFA immunized mice (Fig. 4). InMuSK-immunized mice, NMJ deposit percentages of C3 andMAC were 45.2 ± 8% and 48.3 ± 6%, respectively, whereas CFA-immunized mice did not have any NMJ complement deposits(p b 0.001 by Student's t-test). There were no C3 or MAC posi-tive specimens that were not positive for Ig deposits.
3.4. MuSK immunized mice exhibit increased IL-4and IL-10 production
To investigate the Th1 and Th2 immune responses modulat-ing the IgG1 predominant anti-MuSK antibody productionpattern obtained in three different experiments, the LNCs ofMuSK-CFA and CFA immunized mice were cultured with orwithout MuSK and the levels of the most characteristic Th1(IFN-γ, IL-12), Th2 (IL-4), and regulatory (IL-10) cytokineswere measured in the supernatants of the cultured cells
after 72 h of incubation. Indices for each cytokine werecalculated by taking the ratios of cytokine levels in thesupernatants of MuSK stimulated and non-stimulated LNCsof each mouse. While indices of IL-4, which promotes IgG1production [11,13], and IL-10 were significantly increased inMuSK-CFA-immunized mice as compared to CFA-immunizedmice (p = 0.03 and 0.008 for IL-4 and IL-10 respectively byStudent's t-test), there were no significant differences be-tween two immunization groups in IFN-γ or IL-12 production.Although MuSK-CFA immunized mice showed trends towardsexhibiting higher amounts of IFN-γ, this difference did notattain statistical significance (Fig. 5A). Similar results wereobtained with LNCs cultured for 48 h (data not shown).Similar to the LNC data, the serum levels of IL-4 and IL-10(but not IFN-γ and IL-12) were significantly higher in MuSK-CFA immunized mice than those of CFA immunized mice(p = 0.04 and 0.02 for IL-4 and IL-10 respectively byStudent's t-test, Fig. 5B). MuSK-CFA immunized mice alsoshowed significantly higher serum IL-4/IFN-γ ratios than CFAimmunized mice (p = 0.02 by Student's t-test, Fig. 5C), sug-gesting a possible shift towards Th2-type immune responses.
4. Discussion
The immunogenicity of MuSK is well-established [5]. Similarly,in our experiments, mice immunized with 30 μg of MuSKshowed high (N80%) EAMG incidences and MuSK antibodylevels. Furthermore, we showed that mice immunized withMuSK in CFA and boosted with MuSK in IFA or CFA are equallysusceptible to EAMG and some MuSK-immunized mice mightdevelop muscle weakness in response to a single immuniza-tion. In contrast, for the induction of a high incidence ofEAMG, AChR immunization often requires CFA (containing ad-ditional Mycobacterium butyricum) as an adjuvant duringboosts and EAMG induction with only one AChR immunizationis extremely rare [17,18], suggesting that MuSK might be amore potent immunogen than AChR. Another notable resultwas the considerably greater ability of immunization with30 μg than 10 μg of MuSK to induce EAMG, although thesedoses induced comparable anti-MuSK antibody levels. This
Figure 3 B6 mice immunized with MuSK in CFA (MuSK-CFA) showed progressively increasing EAMG incidence (A), average EAMGgrade (B) and gradually decreasing average grip strength (C) and weight (D) values, whereas mice immunized with CFA only (CFA) didnot develop muscle weakness. Assessment of a broad panel of antibodies in sera (E) and neuromuscular junctions (NMJ) (F) ofimmunized mice showed preferentially increased anti-MuSK IgG1 production and IgG1 deposit accumulation at the NMJ, althoughother Ig isotypes (IgG2b, IgG2c, IgG3 and IgM) were also detected in both serum and muscle samples. The percentages of NMJs withdeposits in each muscle section were calculated by dividing the numbers of Ig deposits by the numbers of bungarotoxin labeled sitesand then multiplying this obtained value with 100. *, p b 0.05; **, p b 0.01; ***, p b 0.001 by Fisher's exact test, Mann–Whitney U andStudent's t-test as required; vertical bars indicate standard errors. One representation of three independent experiments.
160 C. Ulusoy et al.
apparent contradiction might be explained by the productionof low affinity antibodies as a response to immunization withlower amounts of MuSK. Most but not all MuSK antibodypositive MG patients display complement fixing anti-MuSKIgG1 antibodies [7]. However, passive transfer of IgG1 fromMuSK antibody positive patients does not generate muscleweakness in experimental animals whereas injection of IgG4from the same patients readily induces EAMG [4]. This dis-crepancy might also be due to the lower affinity of anti-MuSKIgG1 in humans. Whether only MuSK antibodies with high
affinity can induce myasthenic muscle weakness needs to beexamined by future studies.
As observed in a previous study [19], MuSK immunizationpredominantly induces non-complement fixing anti-MuSK IgG1production. However, the production of much lower levels ofcomplement fixing Ig isotypes (IgG2a, IgG2b in the previousstudy [19] and IgG2b, IgG2c, IgG3, IgM in our study) is alsostimulated by MuSK immunization. This Ig production patternis quite different than the typical Ig production patternof B6 mice. IgG2b is the dominant Ig isotype in B6 serum
Figure 4 Mice immunized with MuSK in CFA (MuSK-CFA) showed IgG, IgG1, IgG2b, IgG2c, IgG3 and IgM deposits, as well as C3 andmembrane attack complex (MAC) deposits, whereas mice immunized with CFA only (CFA) did not show any Ig or complement deposits.Frozenmuscle sections were stained for Ig isotypes, C3 and MAC (bottom panels, all green fluorescence) and the neuromuscular junctions(NMJ) were co-localized by bungarotoxin (top panels, red fluorescence) (magnification for all, ×200). The immunofluorescence datarepresent one of 5 sections for each mouse. One representation of three independent experiments.
161Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice
and similarly, the serum levels of anti-AChR IgG2b in AChR-immunized mice are higher than those of other isotypes[20–22]. Murine IgG1 and human IgG4 are considered to beanalogous because of their biological and functional similari-ties, one of which is failure to activate the complement systemthrough the classical pathway [11,23]. Since anti-MuSK IgG4 isthe dominant isotype in human disease [7], EAMG induced byMuSK immunization appears to closely imitate the immunolog-ical profile of MuSK associated MG.
In line with WT mouse studies, IgG1 KO mice immunizedwith MuSK failed to produce substantial amounts of anti-MuSKIgG at least partially because they lacked the dominant isotype(IgG1) induced by MuSK immunization. In an autoimmune ne-phritis model characterized with antibody and complementmediated renal inflammation, IgG1 KO mice have been shownto be capable of producing complement fixing pathogenic IgG3isotype in high amounts. As a result, IgG1 KO mice are moresusceptible to autoimmune nephritis than WT mice suggestingthat IgG1 has a preventive action in this nephritis model [24].By contrast, in MuSK associated EAMG, IgG1 KO mice showed adeficiency of complement fixing isotype production. Our ex-periments analyzing whether this deficiency contributes toclinical EAMG resistance are in progress. Overall, these resultsindicate a dichotomy in IgG1 functions between complementand non-complement mediated autoimmune diseases. Plausi-bly, based on the autoantigen causing the disease, in someautoimmune disorders (e.g. autoimmune nephritis) com-plement fixing antibodies are generated and IgG1 plays aprotective role. By contrast, in some other autoimmune dis-eases (e.g. MuSK induced EAMG), immunization induces non-complement activating isotypes and therefore IgG1 plays a
pathogenic role. This hypothesis should be further tested bycomparative animal model experiments.
The production of non-complement-fixing Ig isotypes ismainly regulated by Th2-type immunity [IL-4 inmice (IgG1) andIL-4, IL-13 and IL-10 in humans (IgG4)], whereas complement-fixing isotypes (IgG2 and IgG3 inmice, IgG1 and IgG3 in humans)are regulated by Th1-type cytokines (e.g. IFN-γ) [11–13,25].Therefore, increased Th2-type but not Th1-type cytokine pro-duction observed in MuSK-immunized mice is consistent withthe predominance of anti-MuSK IgG1 production. Overall, ourresults suggest that MuSK immunization preferentially stimu-lates the Th2 cytokine IL-4, which subsequently promotesproduction of anti-MuSK IgG1. Nevertheless, marginally in-creased IFN-γ levels in both sera and LNC supernatants and thepresence of complement fixing Ig deposits (IgG2b, IgG2c,IgG3) at the NMJs, albeit at low levels, indicate that Th1-typecytokines are also in play in MuSK immunity. This Th patternclearly differs from AChR immunity, which is mainly regulatedby Th1-type cytokines, while Th2-type cytokines play a lesssignificant role [10]. As a result, antibodies in AChR relatedMGare mostly of the IgG2 isotype in mice and IgG1-3 isotypes inhumans, whereas low levels of anti-AChR IgG1 and IgG4 are alsodetected in mice and patients, respectively [18,21,22,26].
The discrepancy in Th stimulation patterns is naturallynot unique to AChR and MuSK. Previous immunization studieshave shown that molecular and structural properties of theimmunogen and adjuvant are major determinants of the typeof T cell activation. For example, aluminumhydroxide adjuvant(alum) boosts Th2-type immune responses, whereas CFA shiftsthe immune responses towards a Th1 phenotype [27]. As aresult, mild Th1 responses observed in MuSK-immunized mice
Figure 5 Cultured and MuSK- or non-stimulated lymph node cells of mice immunized with MuSK in CFA (MuSK-CFA) showedsignificantly increased average cytokine index values for IL-4 and IL-10 (but not IFN-γ and IL-12) than those of mice immunized withonly CFA (CFA) (A). Cytokine index was calculated for each mouse and cytokine as the ratio between the supernatant level of cytokinemeasured in the MuSK-stimulated culture by the supernatant level of cytokine in the non-stimulated culture (control). Similar tocytokine index data, serum IL-4 and IL-10 (but not IFN-γ and IL-12) levels of MuSK-CFA immunized mice were significantly higher thanthose of CFA-immunized mice (B). Also, Th2/Th1 balance evaluated by the ratio of serum IL-4 and IFN-γ levels were significantlyhigher in MuSK-CFA immunized mice than CFA immunized mice (C). *, p b 0.05; **, p b 0.01 by Student's t-test; vertical bars indicatestandard errors. One representation of three independent experiments.
162 C. Ulusoy et al.
might possibly be due to CFA used as an adjuvant. The sig-nificance of Th1 immunity in MuSK associated MG shouldtherefore be further investigated by EAMG experiments usingnon-Th1 stimulating adjuvants. Whether our results might alsoapply to the so called IgG4-related diseases and these disordersare induced by autoimmunity against Th2 stimulating antigensrequires to be investigated.
Only a small fraction of MuSK antibody positive MG patientsexhibit complement deposits at their NMJs [9], complementdeficient mice are equally susceptible to EAMG induced byMuSK immunization as WT mice [19] and passive transfer ofcomplement fixing isotypes from MuSK antibody positive seradoes not induce EAMG [4]. These results suggest that thecomplement system is not involved in MuSK antibodymediatedpathogenic mechanisms. However, in our experiments sub-stantial amounts of complement deposits were detected atthe NMJs of MuSK immunized mice. One notable aspect of ourstudy was that most of the MuSK immunized mice had verysevere EAMG, suggesting that our model was perhaps animitation of MuSK antibody associated myasthenic crisis.There are only a few muscle pathology reports examiningcomplement deposition in MuSK related EAMG and none ofthem has studied MG patients with myasthenic crisis. Ourresults suggest that complement deposition might plausibly be
involved in myasthenic crisis or severe exacerbations of MuSKantibody positive MG patients.
5. Conclusion
Our results indicate a strong immunogenicity of MuSK.Moreover, IgG1 appears to play differential (suppressive orstimulatory) roles in different autoimmune disorders. Ourfindings also suggest that different to AChR immunity, MuSKimmunity preferentially involves Th2-type responses andsubsequent production of non-complement fixing anti-MuSKantibodies. Potential future cytokine-specific therapies de-signed for AChR related MG might therefore not be beneficialfor MuSK related MG. It is thus imperative to better char-acterize cytokine production patterns in MuSK related MG byconducting cytokine measurements in patients' sera and byperforming MuSK induced EAMG studies using cytokine defi-cient (e.g. IL-4 KO) mice.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
163Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice
Acknowledgments
This study was supported by the Myasthenia Gravis Founda-tion of America, Association Francaise contre les Myopathies,Muscular Dystrophy Association and Hellas–Turkey BilateralProject (109S353) supported by the Scientific and Technolog-ical Research Council of Turkey (TÜBİTAK) and the NSRF“Thalis” Autoimmunity Grant of Greece.
[3] J. Cossins, K. Belaya, K. Zoltowska, I. Koneczny, S. Maxwell, L.Jacobson, et al., The search for new antigenic targets inmyasthenia gravis, Ann. N. Y. Acad. Sci. 1275 (2012) 123–128.
[4] R. Klooster, J.J. Plomp, M.G. Huijbers, E.H. Niks, K.R.Straasheijm, F.J. Detmers, P.W. Hermans, K. Sleijpen, A.Verrips, M. Losen, P. Martinez-Martinez, M.H. De Baets, S.M.van der Maarel, J.J. Verschuuren, Muscle-specific kinase myas-thenia gravis IgG4 autoantibodies cause severe neuromuscularjunction dysfunction in mice, Brain 135 (2012) 1081–1101.
[5] D.P. Richman, K. Nishi, M.J. Ferns, J. Schnier, P. Pytel, R.A.Maselli, M.A. Agius, Animal models of antimuscle-specifickinase myasthenia, Ann. N. Y. Acad. Sci. 1274 (2012) 140–147.
[6] E.H. Niks, Y. van Leeuwen, M.I. Leite, F.W. Dekker, A.R.Wintzen, P.W. Wirtz, A. Vincent, M.J. van Tol, C.M. Jol-van derZijde, J.J. Verschuuren, Clinical fluctuations in MuSK myas-thenia gravis are related to antigen-specific IgG4 instead ofIgG1, J. Neuroimmunol. 195 (2008) 151–156.
[7] M.I. Leite, S. Jacob, S. Viegas, J. Cossins, L. Clover, B.P.Morgan, D. Beeson, N. Willcox, A. Vincent, IgG1 antibodies toacetylcholine receptors in ‘seronegative’ myasthenia gravis,Brain 131 (2008) 1940–1952.
[8] S. Mori, S. Yamada, S. Kubo, J. Chen, S. Matsuda, M. Shudou, N.Maruyama, K. Shigemoto, Divalent and monovalent autoanti-bodies cause dysfunction of MuSK by distinct mechanisms ina rabbitmodel ofmyasthenia gravis, J. Neuroimmunol. 244 (2012)1–7.
[9] H. Shiraishi, M. Motomura, T. Yoshimura, T. Fukudome, T.Fukuda, Y. Nakao, M. Tsujihata, A. Vincent, K. Eguchi, Acetyl-choline receptors loss and postsynaptic damage in MuSK antibody-positive myasthenia gravis, Ann. Neurol. 57 (2005) 289–293.
[10] E. Tüzün, R. Huda, P. Christadoss, Complement and cytokinebased therapeutic strategies inmyasthenia gravis, J. Autoimmun.37 (2011) 136–143.
[11] J. Mestas, C.C. Hughes, Of mice and not men: differencesbetween mouse and human immunology, J. Immunol. 172 (2004)2731–2738.
[12] W. van de Veen, B. Stanic, G. Yaman, M. Wawrzyniak, S. Söllner,D.G. Akdis, B. Rückert, C.A. Akdis, M. Akdis, IgG4 production isconfined to human IL-10-producing regulatory B cells thatsuppress antigen-specific immune responses, J. Allergy Clin.Immunol. 131 (2013) 1204–1212.
[13] A.S. Silva, L.T. Cavalcante, E.L. Faquim-Mauro, M.S. Macedo,Regulation of anaphylactic IgG1 antibody production by IL-4and IL-10, Int. Arch. Allergy Immunol. 141 (2006) 70–78.
[14] S. Jung, K. Rajewsky, A. Radbruch, Shutdown of class switchrecombination by deletion of a switch region control element,Science 259 (1993) 984–987.
[15] N. Trakas, P. Zisimopoulou, S.J. Tzartos, Development of a highlysensitive diagnostic assay for muscle-specific tyrosine kinase(MuSK) autoantibodies in myasthenia gravis, J. Neuroimmunol.240–241 (2011) 79–86.
[16] L. Psaridi-Linardaki, A. Mamalaki, M. Remoundos, S.J. Tzartos,Expression of soluble ligand- and antibody-binding extracellulardomain of human muscle acetylcholine receptor alpha subunit inyeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxinbinding, J. Biol. Chem. 277 (2002) 26980–26986.
[17] B. Wu, E. Goluszko, P. Christadoss, Experimental autoimmunemyasthenia gravis in the mouse, Curr. Protoc. Immunol. 15.8(May 2001) 1–26.
[18] P. Christadoss, M. Poussin, C. Deng, Animalmodels ofmyastheniagravis, Clin. Immunol. 94 (2000) 75–87.
[19] S. Mori, S. Kubo, T. Akiyoshi, S. Yamada, T. Miyazaki, H. Hotta, J.Desaki, M. Kishi, T. Konishi, Y. Nishino, A. Miyazawa, N.Maruyama, K. Shigemoto, Antibodies against muscle-specifickinase impair both presynaptic and postsynaptic functions in amurine model of myasthenia gravis, Am. J. Pathol. 180 (2012)798–810.
[20] A.S. Klein-Schneegans, L. Kuntz, P. Fonteneau, F. Loor, Serumconcentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6mice and their congenics at the lpr (lymphoproliferation)locus, J. Autoimmun. 2 (1989) 869–875.
[21] C. Deng, E. Goluszko, E. Tüzün, H. Yang, P. Christadoss,Resistance to experimental autoimmune myasthenia gravis inIL-6-deficient mice is associated with reduced germinal centerformation and C3 production, J. Immunol. 169 (2002) 1077–1083.
[22] E. Tüzün, B.G. Scott, E. Goluszko, S. Higgs, P. Christadoss,Genetic evidence for involvement of classical complementpathway in induction of experimental autoimmune myastheniagravis, J. Immunol. 171 (2003) 3847–3854.
[23] M.G. Scott, D.E. Briles, M.H. Nahm, Selective IgG subclassexpression: biologic, clinical and functional aspects, in: F. Shakib(Ed.), The Human IgG Subclasses: Molecular Analysis of Structureand Function, Pergamon Press, Oxford, 1990, pp. 161–183.
[24] R. Strait, A. Mahler, N. Barasa, K. Stringer, D. Witte, A. Herr,F. Finkelman, IgG1 protects against IgG3 immune complexmediated renal disease, J. Immunol. 188 (Suppl. 1) (2012) 176.21(Abstract).
[25] T.L. Stevens, A. Bossie, V.M. Sanders, R. Fernandez-Botran, R.L.Coffman, T.R. Mosmann, E.S. Vitetta, Regulation of antibodyisotype secretion by subsets of antigen-specific helper T cells,Nature 334 (1988) 255–258.
[26] A. Rødgaard, F.C. Nielsen, R. Djurup, F. Somnier, S. Gammeltoft,Acetylcholine receptor antibody in myasthenia gravis: predom-inance of IgG subclasses 1 and 3, Clin. Exp. Immunol. 67 (1987)82–88.
[27] D.H. Cribbs, A. Ghochikyan, V. Vasilevko, M. Tran, I. Petrushina,N. Sadzikava, D. Babikyan, P. Kesslak, T. Kieber-Emmons, C.W.Cotman, M.G. Agadjanyan, Adjuvant-dependent modulation ofTh1 and Th2 responses to immunization with beta-amyloid, Int.Immunol. 15 (2003) 505–514.
