Bone marrow chimeric mice were generated to examine TNFR2-/- hematopoietic cells grown in wildtype hosts and and wildtype hematopoietic cells grown in TNFR2-/- hosts.
3.4.1 Reconstitution
Eight weeks after bone marrow transplantation mice were killed and the rates of reconstitution were checked in the spleens. As shown in Figure 40, about 80% reconstitution of the donor bone marrow could be achieved. The distributions of CD4 and CD8 T cells as well as B-cells were not changed in the 4 different groups and were similar to naïve animals.
T cell and B cell reconstitution in bm chimeras
0
Figure 40: Reconstitution of bm chimeric mice
Spleen cell suspensions were prepared and stained according to the congenic markers CD45.1 (A) and CD45.2 (B) of donor and host, respectively. In order to guarantee a natural spleen cell composition, additional stainings for CD4 T cells, CD8 T cells and B cells (B220) were performed and analyzed using flow cytometry (C). Shown are the single values and the mean (horizontal line) of four individual animals per group (A and B) and the mean of the pecentages of the different cells of four individual mice per group (C).
3.4.2 PEC cell distribution and NO production
First, PEC from bm chimeric mice were analyzed for the cellular composition and the NO production capacity. As shown in Figure 41 the cell distributions of PEC in the different chimeras were similar. However, no difference in the NO production could be detected anymore in PEC from wildtype mice that were reconstituted with TNFR2-/- bone marrow.
Figure 41: bm chimeras – PEC distribution and NO production
PEC were generated from bm chimeric mice and analyzed for the distribution of various cell types (A).
Additionally,2.5 x 105 cells were stimulated in 1 mL medium with LPS and IFN-ү (125 ng/mL, 50 ng/mL), in order to measure NO after 48 h (B). Shown are the mean of four individual mice per group (A) and single values and mean (horizontal line) of four individual mice per group (B).
3.4.3 BMDC from bm chimeric mice
BMDC were generated from bm chimeric mice according to the protocol and tested on day 8.
3.4.3.1 Frequency of cells expressing activation markers (MHCII
+CD80
+CD86
+) in BMDC cultures from bm chimeric mice
The frequencies of activated cells in BMDC differentiation cultures of bm chimeric mice expressing the activation markers MHCII+, CD80+, and CD86+ on day 8 are shown in Figure 42.
Significantly increased percentages of mature and activated cells were found in BMDC differentiation cultures of wildtype mice that were reconstituted with TNFR2-/- bone marrow.
These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 24.
bm chimeras PEC NO production
Figure 42: Activation markers of BMDC from bm chimeric mice
The percentages of BMDC expressing the activation markers MHCII, CD80, and CD86 were analyzed employing flow cytometry on day 8. Shown are the single values representing individual mice and the mean (horizontal line) of four different BMDC cultures per group.
3.4.3.2 Nitric Oxide (NO) production of BMDC from bm chimeric mice
Besides, the NO production capacity was investigated in BMDC differentiation cultures of bm chimeric mice on day 8 after stimulation with LPS and IFN-ү. Figure 43 shows that BMDC from wildtype mice reconstituted with TNFR2-/- bone marrow produced significantly reduced amounts of NO. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 13 and Figure 29.
Figure 43: NO production of BMDC from bm chimeric mice
BMDC were removed from the petri dish on day 8 of BMDC differentiation culture. 2.5 x 105 cells were seeded in 1 mL and in 48 well microtiter plates and, subsequently, stimulated with LPS and IFN-ү (125 ng/mL, 50 ng/mL). NO concentrations were measured after 48 h using Griess reagent. Shown are the single values representing individual mice and the mean (horizontal line) of four different BMDC cultures per group.
3.4.3.3 IL-6 production of BMDC cultures from bm chimeric mice
Moreover, the IL-6 production capacity was examined in BMDC differentiation cultures of bm chimeric mice on day 8 after stimulation with LPS and IFN-ү. Figure 44 shows that BMDC from wildtype mice reconstituted with TNFR2-/- bone marrow produced significantly reduced amounts of IL-6. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 30.
bm chimeras BMDC IL-6
0 5 10 15 20 25
Donor: wt TNFR2-/- wt wt
Host: wt wt wt TNFR2 -/-CD45.2 CD45.1
CD45.1 CD45.2 p=0.0008
IL-6 [ng/mL]
Figure 44: IL-6 production of BMDC from bm chimeric mice
BMDC were removed from the petri dish on day 8 of the culture. 2.5 x 105 cells were seeded in 1 mL and in 48 well microtiter plates and, subsequently, stimulated with LPS and IFN-ү (125 ng/mL, 50 ng/mL). IL-6 concentrations were measured after 48 h using ELISA. Shown are the single values representing individual mice and the mean (horizontal line) of four different BMDC cultures per group.
3.4.3.4 sTNF concentrations in BMDC cultures from bm chimeric mice
Additionally, the soluble TNF concentrations were measured in BMDC differentiation cultures of bm chimeric mice on day 8 after stimulation with LPS and IFN-ү. Figure 45 shows that BMDC from wildtype mice reconstituted with TNFR2-/- bone marrow produced slightly increased concentrations of soluble TNF in the supernatant. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 31.
Figure 45: sTNF concentrations in the supernatants of BMDC from bm chimeric mice
BMDC were removed from the petri dish on day 8 of the culture. 2.5 x 105 cells were seeded in 1 mL and in 48 well microtiter plates and, subsequently, stimulated with LPS and IFN-ү (125 ng/mL, 50 ng/mL).
Soluble TNF concentrations were measured after 48 h using ELISA. Shown are the single values representing individual mice and the mean (horizontal line) of four different BMDC cultures per group.
3.4.3.5 sTNFR2 concentrations in BMDC cultures from bm chimeric mice
In parallel, the TNFR2 concentrations were measured in BMDC differentiation cultures of bm chimeric mice on day 8 after stimulation with LPS and IFN-ү. BMDC from wildtype mice reconstituted with TNFR2-/- bone marrow expressed hardly any soluble TNFR2 compared to the wildtype control BMDC indicating almost complete reconstitution. Data are shown in Figure 46.
These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 32.
0 1 2 3 4 5
Donor: wt TNFR2-/- wt wt Host: wt wt wt TNFR2
-/-CD45.2 CD45.1 CD45.1 CD45.2
sTNF [ng/mL]
Figure 46: sTNFR2 concentrations in the supernatants of BMDC from bm chimeric mice
BMDC were removed from the petri dish on day 8 of the culture. 2.5 x 105 cells were seeded in 1 mL and in 48 well microtiter plates and, subsequently, stimulated with LPS and IFN-ү (125 ng/mL, 50 ng/mL).
Soluble TNFR2 concentrations were measured after 48 h using ELISA. Shown are the single values representing individual mice and the mean (horizontal line) of four different BMDC cultures per group.