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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01383

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Ablation of TrkA function in the immune system causes B cell abnormalities

¹ Neural Development Group, Mouse Cancer Genetics Program, NCI, NIH, Frederick, MD 21702-1201, USA
² Experimental Immunology Branch, NCI, NIH, Bethesda, MD 20892, USA
³ Aventis Pharmaceuticals, Bridgewater, NJ 08807-0800, USA

View larger version (57K): [in a new window]   Fig. 1. Conditional inactivation of TrkA. (A) Diagram of the replacement vector strategy used to generate the TrkAneo allele and re-activation of TrkA expression by Cre recombinase. The pGKneobpA cassette flanked by loxP sites was placed into intron 11. (B) Southern blot analysis of tail DNA from three-week-old mice with a 5' external probe shows the switch of the wild-type (WT) BamHI fragment from 12 kb (WT) to a 7 kb restriction fragment (TrkAneo) after neo insertion. (C) Northern blot analysis of total RNA extracted from Dorsal Root Ganglia (DRG) neurons of E13.5 embryos either WT (+/+), heterozygous for the neo insertion (+/neo), homozygous for the neo insertion (neo/neo), or homozygous for the allele after neo removal with ß-actin cre (loxP/loxP), hybridized with a TrkA-kinase domain-specific probe (exon 14-17). Note that neo insertion completely eliminates TrkA-specific transcripts whereas TrkA-transcription is restored to WT levels after neo-excision by Cre recombination. (D) RT-PCR analysis of the same samples analyzed by northern blot. Specific primers for actin, neo, and TrkA exon 10 and 14 were used. Samples were treated with (+) and without (–) RT. Note the complete absence of the 609 bp TrkA-specific PCR product in TrkAneo/neo mice. (E) Wholemount lacZ staining of a Rosa-26 E11.5 embryo with the T1-cre transgene. Note the specific staining in both central and peripheral nervous system. (F) lacZ staining of sections from spleen, thymus and bone marrow of adult Rosa-26 mice carrying the T1-Cre (top panels) or a ß-actin-Cre transgene with mosaic expression used as control (bottom panels) (Ma et al., 2003). Cre recombinase under the control of the T-1 promoter is only expressed in the nervous system (E) and not in organs of the immune system (F).

View larger version (75K): [in a new window]   Fig. 2. TrkA deficiency does not affect spleen development and skin mast cells. Spleens of wild-type (A,C,E,G) and mutant (B,D,F,H) mice were stained with hematoxylin/eosin (A,B), Apoptag (C,D), and B cell- (anti-B220; E,F) and T cell-specific (anti-CD3; G,H) antibodies. Note the absence of any remarkable difference between mutant and control organs. (L,M) Toluidine-blue staining of mutant (M) skin samples shows presence of mast cells (arrows) in number and distribution similar to wild-type littermate controls (L). LF, lymphoid follicle.

View larger version (87K): [in a new window]   Fig. 3. Mast cells degranulate in TrkA-deficient mice. Histological assessment of passive cutaneous anaphylaxis in control (A,C,E) and TrkA mutant (B,D,F) mouse ear sections stained for naphthol AS-D chloroacetate esterase. Compared with saline challenged mice (A,B), the IgE/DNP-treated mice (C-F) exhibited prominent mast cell degranulation and mixed inflammatory cell infiltrates. Arrows are directed to degranulated mast cells. (G) ß-hexoseaminidase release from bone marrow-derived mast cells obtained from TrkA mutant (TrkAneo/neo) and control (TrkA+/+) littermate mice was measured as described in Materials and methods. Mast cells obtained from the TrkA mutant mice show reduced ß-hexoseaminidase release relative to those from controls (*P<0.01).

View larger version (16K): [in a new window]   Fig. 4. TrkA deficiency in the immune system causes increased Ig production. (A) T1-Cre;TrkAneo/neo mice have increased levels of specific classes of Igs. IgM, IgG2a, IgG2b, IgG1 and IgG3 serum levels were measured by ELISA in 12-16-week-old mutants (KO, n=18) and control littermates (CTR, n=14). Data are expressed in Units/ml where 1 U=1 mg for IgM and 1 U=1 µg for IgG2a, IgG2b, IgG1 and IgG3. Bars: means±s.e.m. Data were evaluated using a two-tailed Student's t-test. *P<0.05. (B) C57Bl/6-SCID mice reconstituted with TrkA-deficient fetal liver cells (KO, n=10) have increased levels of serum Igs compared with TrkA wild-type-reconstituted controls (WT, n=6). C57Bl/6-SCID mice were reconstituted and monitored for Ig serum levels from 1 to 5 months after reconstitution. Data after two months from reconstitution are shown. All Ig classes are higher in TrkA-deficient animals, although only IgM and IgG2b are statistically different from controls (two-tailed Student's t-test. *P<0.05).

View larger version (19K): [in a new window]   Fig. 5. Absence of TrkA does not impair T-cell-dependent memory nor T-cell-independent type I and II B responses. (A) For the TD memory response, T1-Cre;TrkAneo/neo (n=7) and control littermates (n=6) were immunized with KLH-TNP as described in Materials and methods. (B,C) for the TI type I and II responses, mice were immunized respectively with LPS-TNP (B, six mutants and five controls) or Ficoll-TNP (C, six mutants and seven controls). Serum levels of specific anti-TNP Igs were measured by ELISA before immunization (0), at one (1) and two (2) weeks after the challenge. Bars: means±s.e.m. Data were evaluated using a two-tailed Student's t-test. *P<0.05.

View larger version (49K): [in a new window]   Fig. 6. Aged T1-Cre;TrkAneo/neo mice have increased levels of peritoneal B1 cells. Twelve- to eighteen-month-old mutant (n=6) and control (n=6) littermates were sacrificed and peritoneal cells were collected as previously described (32). Similar numbers of cells were recovered from both groups. Cells were stained for FACS analysis with anti-B220, -IgM, -CD5, IgD and -Mac-1 antibodies in triple staining combinations. Three staining combinations are shown (B220/IgM; Mac-1/IgD; B220/CD5). The B1 cell population is indicated by a box. Percentages of peritoneal B2 cells (B220+/IgMlow/IgDhigh/Mac-1–) and macrophages (B220–/Mac-1+) were not different in mutant mice compared with controls.

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