Impact of mono-culture vs. Co-culture of keratinocytes and monocytes on cytokine responses induced by important skin sensitizers

Figures & data

Figure 1. Cell viability. Viability was determined by an Alamar blue assay after 24-h exposure of three cell culture systems (HaCaT or THP-1 mono-cultures and HaCaT/THP-1 co-culture) with different concentraions (µg/ml) of CoCl₂, MI, and PPD. Each bar represents mean ± SE of four experiments. Statistical analysis: one-way ANOVA or Bonferroni’s multiple comparison. Value significantly different from unexposed/control cells, ***p ≤ 0.001.

Figure 2. Effect of CoCl₂ on secretion of pro-inflammatory cytokines in the three culture systems. IC₂₀ concentrations used; HaCaT= 117.5, THP-1 = 128.7 and co-culture= 101.5 µg/ml. Assessment of cytokines was performed after 24 h of exposure. Each bar (n = 4, mean ± SE) represents the concentration of the mediator (pg/ml). Statistical analysis: one-way ANOVA or Bonferroni’s multiple comparison. Value significantly different from corresponding control: *p ≤ 0.05, **p ≤ 0.01.

Figure 3. Effect of MI on secretion of pro-inflammatory cytokines in the three culture systems. IC₂₀ concentrations used; HaCaT = 25.1, THP-1 = 16.5 and co-culture = 17 µg/ml. Assessment of cytokines was performed after 24 h of exposure. Each bar (n = 4, mean ± SE) represents the concentration of the mediator (pg/ml). Statistical analysis: one-way ANOVA or Bonferroni’s multiple comparison. Value significantly different from corresponding control: *p ≤ 0.05, **p ≤ 0.01.

Figure 4. Effect of PPD on secretion of pro-inflammatory cytokines in the three culture systems. IC₂₀ concentrations used; HaCaT = 15.0, THP-1 = 38.0 and co-culture= 77.7 µg/ml. Assessment of cytokines was performed after 24 h of exposure. Each bar (n = 4, mean ± SE) represents the concentration of the mediator (pg/ml). Statistical analysis: one-way ANOVA or Bonferroni’s multiple comparison. Value significantly different from corresponding control: *p ≤ 0.05, **p ≤ 0.01.

Table 1. Summary of cytokine responses.

Assay	IFNγ	IL-10	IL-12p70	IL-13	IL-1β	IL-2	IL-4	IL-6	IL-8	TNFα
CoCl2
HaCaT	1.1	1.1	1.2	0.8	1.1	1.3	1.4	0.9	1.3	1.1
THP 1	1.1	1.2	1.1	6.4	1.5	1.7	0.9	4.0	13.5	0.6
Co-culture	1.0	0.1	0.4	6.4	7.6	1.0	1.5	0.7	10.5	0.4
MI
HaCaT	2.0	9.9	23.0	2.5	11.7	(3.2)	(6.3)	5.8	0.2	15.9
THP 1	0.9	(4.1)	1.2	(2.6)	4.0	(2.1)	1.3	(2.3)	3.7	2.1
Co-culture	1.7	0.2	0.7	(2.7)	48.7	1.0	1.7	8.6	6.3	2.6
PPD
HaCaT	1.5	(2.3)	1.4	1.2	8.5	(3.2)	(2.4)	1.2	0.6	4.7
THP 1	1.1	(2.6)	0.9	1.1	2.2	(2.9)	0.8	0.8	2.4	1.1
Co-culture	1.0	0.0	0.4	(2.1)	3.8	1.1	1.0	0.5	16.2	1.1

Data expressed as “stimulation index” (SI) in mono- and co-cultures exposed to CoCl₂, MI, and PPD. SI in parentheses indicate changes were over the threshold (SI > 2), but were not statistically significant.

Shade gradient (low to high) indicates the SI values range (>2.0 – < 3.0, 3.0 – 10.0, and ≥ 10, respectively).

Table 2. Summary of cytokine responses to control agents.

Co-culture Assay	IFNγ	IL-10	IL-12p70	IL-13	IL-1β	IL-2	IL-4	IL-6	IL-8	TNF-α
LA	5.2	0.4	ND	0.7	0.2	1.3	ND	0.4	0.3	0.4
SDS	2.4	0.2	ND	1.8	1.1	1.4	ND	1.2	0.3	1.1

Data are expressed as “stimulation index” (SI) in co-cultures exposed to the negative controls LA (IC₂₀ >1000 µg/ml) and SDS (IC₂₀ 14.6 µg/ml).

SI values and shade gradient are explained in Table 1.

ND: Reported as “no data” due to values being too low for quantification.