R-Ras deficiency does not affect papain-induced IgE production in mice

Abstract

Introduction: R-Ras GTPase has recently been implicated in the regulation of immune functions, particularly in dendritic cell (DC) maturation, immune synapse formation, and subsequent T cell responses.

Methods: Here, we investigated the role of R-Ras in allergen-induced immune response (type 2 immune response) in Rras deficient (R-Ras KO) and wild type (WT) mice.

Results: Initially, we found that the number of conventional DC's in the lymph nodes (LNs) was reduced in R-Ras KO mice. The expression of co-stimulatory CD80 and CD86 molecules on these cells was also reduced on DC's from the R-Ras KO mice. However, there was no difference in papain-induced immune response between the R-Ras WT and KO as measured by serum IgE levels after the immunization. Interestingly, neither the DC number nor co-stimulatory molecule expression was different between WT and R-Ras KO animals after the immunization.

Conclusions: Taken together, despite having reduced number of conventional DC's in the R-Ras KO mice and low expression of CD80 on DC's, the R-Ras KO mice are capable of mounting papain-induced IgE responses comparable to that of the WT mice. To our knowledge, this is the first report addressing potential differences in in vivo allergen responses regulated by the R-Ras GTPase.

Keywords: Allergology; IgE; R-Ras; dendritic cells; knock-out mice.

Figure 1

Immunophenotyping of un‐manipulated R‐Ras KO mice. (A) Lymphocyte populations of “off‐the‐shelf” wild type (WT, n = 3) or R‐Ras deficient (R‐Ras KO, n = 3) mice were compared by flow cytometry. (B and C) LNs (B) and spleens (C) were collected from untreated wild type (WT, n = 3) or R‐Ras deficient (R‐Ras KO, n = 3) mice. LNs from each mouse were pooled separately. DC's were enriched by magnetic separation (LN) or gradient centrifugation (spleens) and analyzed by flow cytometer. LN MHCII + CD11c+ (DC all) cell group includes two populations: conventional (convDC) and migratory dendritic cells (migrDC). (B) The amount of all MHCII + CD11c + dendritic cells (DC all) and their distribution between conventional (convDC) and migratory (migrDC) dendritic cells. (C) The amount of MHCII + CD11c + dendritic cells in the spleen. For statistical analysis unpaired two‐tailed t‐tests were used. *p < 0.05.

Figure 2

Comparison of the expression of dendritic cell surface proteins and expression of T‐helper cell transcription factors and cytokines between WT and R‐Ras KO mice. LNs (A) and spleens (B) were collected from untreated wild type (WT, n = 3) or R‐Ras deficient (R‐Ras KO, n = 3) mice and DC's were enriched as in Figure 1. (A) The expression of MHCII, CD80, OX40L, and CD86 surface proteins in conventional (convDC) and migratory dendritic cells (migrDC) from the LNs and (B) from the spleen. (C) Spleens of un‐manipulated WT and R‐Ras KO mice (n = 4 for each genotype) were harvested. mRNA was isolated from total splenocytes and transcribed into cDNA. The expression of Th‐specific transcription factors and cytokines were investigated with qPCR. The differences within surface protein expression (A and B) or gene expression (C) values were statistically analyzed by two‐tailed unpaired t‐tests. *p < 0.05, **p < 0.01. Each dot represents an individual animal. Mean values with SEM are marked in the scatter dot plots.

Figure 3

The effect of papain‐immunization on WT and R‐Ras‐deficient mice. Wild type (WT) or R‐Ras deficient (R‐Ras KO) mice were treated with papain (500 μg/animal, subcutaneous injection on day 0 and day 14). Animals were euthanized either on day 21 or day 16. IgE concentration of blood serum was measured using ELISA on (A) day 21 or (B) day 16. Experiment in A was done twice with four mice of each genotype and in B the experiment was done four times with 4–5 mice of each genotype. (C and D) LNs (n = 4) and spleens (n = 5) were collected from papain‐immunized (day 16) wild type (WT) or R‐Ras deficient (R‐Ras KO) mice and dendritic cells were enriched as in Figure 1. (C) The amount of all LN MHCII + CD11c + dendritic cells (DC all) and their distribution between conventional (convDC) and migratory (migrDc). (D) The amount of MHCII + CD11c + dendritic cells in the spleen. (E) Expression of IL‐4Rα in CD4 and CD8 T cells of WT and R‐Ras KO mice (n = 5 each genotype) on day 16 after papain immunization. (F) Percent of IL‐33R positive cells of CD4 T cells from WT and R‐Ras KO mice on day 16 after papain immunization. Mean values with SEM are marked in the scatter dot plots. Each dot represents an individual animal. *p < 0.05 (unpaired t‐tests).

Figure 4

The expression of dendritic cell surface markers and mRNA expression of T‐helper cell transcription factors and cytokines after papain‐immunization in WT and R‐Ras KO mice. (A) LNs were collected from papain‐immunized (day 16) wild type (WT, n = 4) or R‐Ras deficient (R‐Ras KO, n = 4) mice and dendritic cells were enriched by magnetic separation. The expression of different surface proteins (MHCII, CD80, OX40L, and CD86) was investigated directly after euthanizing the mice. (B) Spleens were collected from papain‐immunized (day 16) wild type (WT, n = 5) or R‐Ras deficient (R‐Ras KO, n = 5) mice and dendritic cells were enriched by gradient centrifugation. The expression of different surface proteins (MHCII, CD80, and CD86) was analyzed with flow cytometer. The differences within surface protein expression were statistically analyzed (unpaired two‐tailed t‐tests. *p < 0.05). (C) Spleens were harvested from day 16 papain‐immunized wild type (WT, n = 5) and RAS‐deficient (R‐Ras KO, n = 5) mice and prepared as in Figure 2. qPCR analysis for T cell transcription factors and cytokines was performed. Each dot represents an individual animal. Mean values with SEM are marked in the scatter dot plots. The differences within gene expression values were statistically analyzed by two‐tailed unpaired t‐tests. *p < 0.05