Effect of WT, Nf1+/–, and Nf1–/– Schwann cell CM on WT and Nf1+/– mast cell haptotaxis. (a) Transwells were coated with recombinant FN and mast cell migration assays were performed in response to WT, Nf1+/–, and Nf1–/– Schwann cell CM. The number of WT and Nf1+/– mast cells that had migrated to the bottom surface of the FN-coated membrane in response to Schwann cell CM were counted after staining the cells with crystal violet. A representative photomicrograph of the WT- and Nf1+/–-migrated mast cells, which stain purple, is shown for each experimental condition. (b) The average numbers of WT and Nf1+/– mast cells per ten high-power fields, which migrated in response to either 105 WT, Nf1+/–, or Nf1–/– Schwann cell CM are shown. Data represent the mean number of migrated cells per ten high-power fields ± SEM of four independent experiments. *P < 0.05 for Nf1+/– versus WT mast cells in response to either WT Schwann cell CM or Nf1+/– Schwann cell CM. **P < 0.05 for WT mast cells in response to both WT and Nf1+/– Schwann cell CM versus Nf1–/– Schwann cell CM; ***P < 0.01 for Nf1+/– mast cells in response to both WT and Nf1+/– versus Nf1–/– Schwann cell CM by the Student’s paired t test. HPF, high-power field.

Effect of heterozygosity of Nf1 on mast cell haptotaxis and F-actin content in response to rmKitL. (a) Haptotaxis of WT and Nf1+/– mast cells in response to 10 ng/ml rmKitL. Results represent the mean ± SEM of five independent experiments. *P < 0.05 for Nf1+/– versus WT by the Student’s paired t test. (b) Sequential exposures of Nf1+/– and WT mast cells taken at 2.5-minute intervals during exposure to a 10 ng/ml KitL gradient (from left to right) for 1 hour. Mast cells were allowed to adhere to FN-coated glass coverslips for 15 minutes before being loaded into the chemotactic gradient of a Zigmond chamber. Nf1+/– mast cells demonstrated greatly enhanced movement to the rmKitL gradient compared with WT cells. (c) Percentage of Nf1+/– or WT cells moving at various speeds during videomicroscopy. The average speed of directed movement was calculated from data collected from the second 30-minute interval of videomicroscopy from WT and Nf1+/– samples (n >100 cells analyzed per genotype). Histogram depicts the results from one representative experiment. Two other experiments showed similar results. (d) F-actin content in BMMCs. Mast cells were stimulated with 10 ng/ml rmKitL and fixed, at the time points indicated, by addition of formaldehyde. WT and Nf1+/– cells were examined in triplicate. Results are expressed as mean channel fluorescence (MCF) and represent the mean of four independent experiments per genotype. *P < 0.001 for all comparisons between genotypes using a Student’s paired t test.

Haptotaxis of Nf1+/– mast cells expressing NF1 GRD or R1276P mutant NF1 GRD. Nf1+/– mast cells were transduced with the control retrovirus (pac), NF1 GRD virus, or R1276P NF1 GRD virus, and haptotaxis assays were performed in response to rmKitL. Results represent the mean ± SEM of three independent experiments. *P < 0.05 for comparison of Nf1+/– pac and Nf1+/– R1276P NF1 GRD to WT pac using a Student’s paired t test.

Isolation of purified populations of Schwann cells from day 13.5 WT and Nf1–/– embryonic DRG. Immunohistochemical staining of S-100 Schwann cells.

Quantification of the concentration of KitL in Schwann cell CM and the effect of pharmacologic or genetic inhibition of c-kit activity on mast cell migration to Schwann cell CM. (a) Concentration of murine KitL in WT, Nf1+/–, and Nf1–/– Schwann cell CM was determined by ELISA. Results represent the mean ± SEM of five independent collections from five independent Schwann cell cultures. *P < 0.05 for Nf1–/– versus WT or Nf1–/– versus Nf1+/– by the Student’s paired t test. (b) WT and Nf1+/– mast cells (2 × 105) were preincubated with a neutralizing Ab to the c-kit RTK, and haptotaxis assays were performed to either WT or Nf1–/– Schwann cell CM. Results represent the mean ± SEM of five independent experiments. *P < 0.05 for WT mast cell migration in response to WT or Nf1–/– Schwann cell CM in the presence or absence of the c-kit–neutralizing Ab. **P < 0.05 for Nf1+/– mast cell migration in response to WT or Nf1–/– Schwann cell CM in the presence or absence of the c-kit–neutralizing Ab by the Student’s paired t test. (c) Haptotaxis of mast cells of the four Nf1 and W genotypes in response to either 2 × 105 WT or Nf1–/– Schwann cell CM. Results represent the mean ± SEM of five independent experiments. *P < 0.05 for W41/W41 versus WT mast cell migration in response to either WT or Nf1–/– Schwann cell CM. **P < 0.05 for Nf1+/–; W41/W41 versus Nf1+/– mast cell migration in response to either WT or Nf1–/– Schwann cell CM by the Student’s paired t test.

Effect of inhibition of adherence to the α₄β₁or α₅β₁ integrins on WT and Nf1+/– mast cell haptotaxis. WT and Nf1+/– mast cells were preincubated with a neutralizing Ab to the α₄β₁ or α₅β₁ integrins, or an isotype control Ab, and haptotaxis assays were performed to rmKitL. Results represent the mean ± SEM of five independent experiments following a 3-hour stimulation to KitL. *P < 0.05 for WT or Nf1+/– mast cell migration in response to recombinant KitL in presence or absence of the α₄β₁-neutralizing Ab or the isotype Ab control using a Student’s paired t test.

Effect of Rac2 or p85α deficiency on the haptotaxis of Nf1+/– and WT mast cells. (a) Haptotaxis of mast cells of the four Nf1 and p85α genotypes in response to rmKitL. *P < 0.01 for WT versus Nf1+/–. **P < 0.01 for WT versus p85α–/–. ***P < 0.01 for Nf1+/– versus Nf1+/–; p85α–/– by the Student’s paired t test. (b) Haptotaxis of mast cells of the four Nf1 and Rac2 genotypes in response to rmKitL. *P < 0.01 for WT versus Nf1+/–.; Nf1+/+. **P < 0.01 for Nf1+/–-; Nf1+/+ versus Nf1+/–; Rac2–/–. ***P < 0.01 for WT versus Rac2–/– by the Student’s paired t test.