Beta common receptor inactivation attenuates myeloproliferative disease in Nf1 mutant mice

Abstract

Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor, which encodes neurofibromin, a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-, beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly, when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells, concomitant loss of beta c-/- reduced the severity of the MPD, but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML, our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor.

Figure 1

Experimental design of transplants. (A) Three classes of embryos result from the genetic crosses. X¹ is an outcross of Nf1^{+/− -}to β_c^−/− mice. This is to establish Nf1 and β_c null alleles within the same animal. X² is another outcross to β_c^−/− mice to homozygose the β_c allele in Nf1^+/−, β_c^−/− mice. By intercrossing, Nf1^+/−, β_c^−/− mice X³, the 2 classes of embryos are generated. (B) To test the effect loss of β_c has on Nf1^−/−-induced MPD, Nf1^+/−, β_c^−/− mice are intercrossed and fetal livers from embryos of the correct genotype are harvested. Lethally irradiated recipients are injected with 2 million fetal liver cells and allowed a 4- to 8-week recovery period. These primary recipients are then killed to provide bone marrow for secondary recipient transplantation.

Figure 2

Engraftment but lack of MPD in Nf1^−/−, beta c^−/− recipients. (A) Bone marrow from 3 representative irradiated mice given transplants with Nf1^−/−, beta c^−/− (top panel) and Nf1^+/−, beta c^−/− (bottom panel) cells was mostly comprised of cells expressing donor CD45.2, with only a few cells staining positive for CD45.1. Recipients that do not receive donor cells do not stain for the donor-cell marker (data not shown). (B) Flow cytometric analysis of 2 representative mice. Bone marrow and peripheral blood stained for the cell-surface markers Gr-1 and Mac-1, markers commonly associated with mature granulocytes. Similar staining profiles were seen in all recipients. Ranges of double-positive Gr-1/Mac-1 cells in both cohorts, bone marrow 52% to 68% and peripheral blood 12% to 20%. (C) Peripheral-blood analysis monitored over 46 weeks Nf1^+/−, beta c^−/−and Nf1^−/−, beta c^−/− secondary recipients. Animals that display symptoms of MPD would show drastic increases in total WBC counts outside of the range indicated by the bar. Left graph illustrates peripheral-blood analysis monitored over 56 weeks Nf1^+/−, beta c^−/− and Nf1^−/−, beta c^−/− secondary recipients. Upper lines are lymphocyte percentages and lower lines are neutrophil percentages. Animals that display symptoms of MPD would show drastic increases in neutrophil percentages and reductions in lymphocytes, with values outside of the normal ranges indicated by the dotted (lymphocyte) and solid (neutrophil) bars. Four data points included for Nf1^+/+, beta c^+/+ primary recipients to illustrate normal values.

Figure 3

Recipients of Nf1^−/−β_c^−/− bone marrow have increased CFCs and CFU-Ss. (A) Spleen and bone marrow cells from 5 secondary mice given transplants with Nf1^−/−, β_c^−/− or 4 secondary mice given transplants with Nf1^+/−, β_c^−/− fetal stem cells were assayed for CFU-GMs, -Gs, -Ms, and -GEMMs and erythroid burst-forming units. Spleen cells from animals given transplants do not show a significant difference from sample to sample, P = .164. Bone marrow plated from 5 mice given transplants with Nf1^−/−, β_c^−/−or Nf1^+/−, β_c^−/− fetal stem cells have a significantly higher number of cells capable of colony formation than the Nf1^+/−, β_c^−/− transplants, P < .001. (B) Spleen and bone marrow cells from 6 mice given transplants with Nf1^−/−, β_c^−/−or 9 mice given transplants with Nf1^+/−, β_c^−/− fetal stem cells were injected intravenously into mice irradiated with 750 rads to assay for CFU-Ss. Injected spleen cells do not show a significant difference from sample to sample, P = .228. Bone marrow cells in animals given Nf1^−/−, β_c^−/− transplants have a significantly higher number of CFU-S progenitor cells than the Nf1^+/−, β_c^−/− transplants, P < .001. Error bars indicate SE.

Figure 4

Effects of beta c genotype on leukocyte counts in Mx1-Cre, Nf1^flox/flox mice. (A) Data from 6 Mx1-Cre, Nf1^flox/flox, beta c^−/−(○) and 6 Mx1-Cre, Nf1^flox/flox, beta βc^+/+ mice (▵) show a modest elevation in total leukocyte counts relative to 5 beta c^−/−(●) or 7 wild-type (▴) animals. Leukocyte counts are significantly higher in the Mx1-Cre, Nf1^flox/flox, beta c^+/+ group by 6 months of age. (B) Lymphoid (□) and myeloid (■) cell counts are shown at 3 months of age for mice of each genotype: 12 Mx1-Cre, Nf1^flox/flox, beta c^+/+; 12 Mx1-Cre, Nf1^flox/flox, beta c^+/−; 8 Mx1-Cre, Nf1^flox/flox, beta c^−/−; 8 beta c^+/+; 12 beta c^+/−; 9 beta c^−/−. Error bars indicate SD from the mean.

Figure 5

Splenic infiltration in Mx1-Cre, Nf1^flox/flox, beta c^−/− and Mx1-Cre, Nf1^flox/flox, beta c^+/+ mice. (A) Inactivation of Nf1 leads to increased numbers of myeloid lineage cells in spleen and liver, which is most pronounced in 6 Mx1-Cre, Nf1^flox/flox, beta c^+/+ animals and absent in 7 wild-type and 5 beta c^−/− spleens. (B) Spleen weights in 7 control and 6 Mx1-Cre, Nf1^flox/flox mice. Loss of Nf1 results in splenomegaly and is attenuated 6 Mx1-Cre, Nf1^flox/flox, beta c^−/− mice. (C) CFU-GM colony numbers from 4 individual experiments of mice of all 4 genotypes demonstrate infiltration in the Nf1 mutant animals.