INVERSOR

Since the in vivo effects of cytokines on MK production and migration may depend on the duration and timing of exposure to these factors, experiments were performed in three different ways in an attempt to reproduce these variables. In the first set of experiments CD34^ cultures were grown in the presence of either IL-6, IL-II or TPO

alone (Figure 9.9A) for a period of 12 days to establish the direct effects these cytokines may have on proliferation of the culture as a whole. In the second set of experiments the CD34^ cultures were all grown in the presence of TPO to which either IL- 6 or IL-11 were added to see whether there was a synergistic effect on

proliferation between TPO and these cytokines (Figure 9.9B). As shown in Figure 9.9A, neither the presence of IL- 6 or IL-I 1 in the cultures is by itself able to induce

proliferation of the CD34^ cultures. TPO by itself lead to a 14-fold expansion of cells within these cultures. A small synergistic effect was seen between TPO and IL-11 in the expansion of these cultures (Figure 9.9B). The effect of TPO on expansion of these cultures would appear to be maximal between days 6 and 1 2.

15- 2 1 0- <u .o E 3 C ^ 5 O mm TPO □ IL-6 □ IL-11

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a

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Figure 9.9 Expansion of CD34^ derived cultures expressed relative to number of cells seeded on day 1.

A Cultures grown in the presence of either TPO 20ng/mL, IL- 6 lOng/mL or IL-I I

50ng/mL only. B All cultures grown in the presence of TPO 20ng/mL alone or with the addition of IL- 6 lOng/mL or IL-I I 50ng/mL at the time of seeding. Results are

These preliminary experiments established that since IL- 6 and IL-11 by themselves

were not capable of expanding the CD34^ cultures, further experiments to test the effects of these cytokines on MK production and migration would also require the addition of TPO to all the cultures from the beginning. Experiments were performed in two ways in order to model the effects of exposure of the cultures to the cytokines from day 1 and also to examine the effects of a sudden exposure to these cytokines at

a later maturational stage (day 8). These data are presented in Tables 9.4 & 9.5.

Table 9.4 shows the results of the addition of all cytokines to the CD34^ cultures on day 1 whereas Table 9.5 shows the results of growing all cultures in TPO only for the first 8 days and then adding IL-6, IL-11 or additional TPO at day 8.

Table 9.4 MK production in single compartmental system with early cytokine exposure Treatment Total cells (xlCf) p count relative to TPO only %MKs No. MKs (x l0 ‘) pM K relative to TPO only Ploidy o f MKs (%) 2n >4n TPO 2.94 ± 0.11 61 ± 0.6 1.82 ± 0 .0 1 4 65 35 TPO/IL-6 3.22 ± 0 .1 4 ns 60 ± 0.6 1.96 ± 0 .0 5 6 ns 63 37 TPO/IL-11 4.06 ± 0 .5 6 ns 64 ± 0.7 2.66 ± 0.07 ns 63 37

Effects of TPO (20ng/ml) alone or with either IL- 6 (20ng/ml) or IL-11 (50ng/ml)

added on day 0 on the production and characteristics of CD34^ derived MKs after 12 days in culture. Results are the mean ± s.e.m. of 3 experiments.

Table 9.5 MK production in single compartmental system with late cytokine exposure

Treatment

Total cells/ml p count relative to TPO only %MKs No. MKs pM K relative to TPO only Ploidy o f MKs /0 /\ (x l0 ‘) (xlO®) 2n >4n TPO 5.6 ± 0 .5 6 75 ± 0 4.2 ± 0.42 75 25 TPO + IL-6 3.64 ± 0 .2 8 0.04 63 ± 0.6 1.82 ± 0 .1 4 0.03 62 38 TPO + IL-11 3.22 ± 0.56 0.04 59 ± 4.0 1.96 ± 0 .4 2 0.03 61 39

Effects of addition of TPO (20ng/ml), IL- 6 (20ng/ml) or IL-11 (50 ng/ml) to CD34^

derived cultures grown in TPO at day 8 on MK characteristics measured at day 12.

Interestingly, the proliferation and differentiation of MKs within cultures exposed to the different cytokines from day 1 was similar with no statistical differences found

between total numbers of cells, total number of MKs produced and nuclear ploidy distribution of the MKs (table 9.4).

However when the cytokines were added to the culture at day 8 significant

differences between total number of cells and MKs produced by the cultures were seen (table 9.5). Cultures exposed solely to TPO underwent greater expansion in total cell numbers and increased production of MKs compared to cultures that had IL- 6 or

IL-11 added. However the addition of TPO at day 8 also resulted in the production of

cultures with significantly lower proportions of differentiated cells (25 ± 0.4 % MKs with ploidy >4n in TPO vs. 38 ± 0.6 and 39 ± 1.6 % respectively in TPO + IL- 6 and

TPO 4- IL-11, p=0.0002 and 0.009 respectively).

Also when compared to cultures that had only been treated with TPO on day 0 (table 9.4) there was a significant increase in the number of MKs produced by these cultures (1.82 ± 0.014 x 10^ MKs in cultures treated only on day 1 vs. 4.2 ± 0.42 x 10^ MKs when TPO was supplemented on day 8, p<0.05). This proportional loss in

higher ploidy cells was also seen when compared to cultures that had only received TPO on day 0 (35 ± 3.4 % in TPO day 0 vs. 25 ± 0.4% in TPO supplemented day 8)

although this difference did not reach statistical significance (p=0.08).

Having established that the timing of the addition of cytokines to the CD34^ cultures affected the characteristics of the cultures, experiments were performed in two ways to test the effects of these factors on migration of MKs in the bicompartmental system in the presence of an endothelial cell monolayer. In the first set of experiments, CD34^ cultures were grown from day 0 in the presence of the TPO alone or TPO with IL- 6 or IL-11 added. At day 8 the cells were washed and seeded

into the top well of the bicompartmental system in cytokine-free medium (MK medium A). Cells in the top and bottom wells were counted at day 12 i.e. after 4 days in the bicompartmental system. No differences were seen either in the overall percentage migration of MKs or the ploidy of the migrating cells between the different cytokine combinations (data not shown).

In order to establish whether TPO, IL- 6 or IL-11 may have any direct effects on

migration, a second set of experiments were performed in which CD34^ cultures were grown in the presence of TPO only for a period of 12 days before seeding into the top well of the bicompartmental system. Experiments in which TPO, IL- 6 and IL-

11 were then added to top and bottom chambers for a 24 h migration period failed to show significant differences in the percentage of MKs migrating (data not shown).