A set of microphotographs and a graph documenting inhibition of neutrophil migration towards 10 µM CuSO₄ exposed hair cells resulting from co-treatment of 5 dpf Tg(MPX:GFP) zebrafish larvae (exhibiting green fluorescence in neutrophils) with 100 nM PACAP-38.

<p>(A) The control untreated larva presented normal distribution of neutrophils which were found in the ventral myotomes of the trunk and tail. (B) 10 µM CuSO₄ exposure evoked the migration of the immune cells towards the midline of the body and the formation of characteristic concentrations very close to, and around, the neuromasts (arrows). (C) 100 nM PACAP-38 co-treatment resulted in the inhibition of the neutrophil migration, which was reflected by a decreased number, or complete lack of, the green fluorescent cells in the area of natural neuromast localization (arrows). (D) 100 nM PACAP-38 itself did not visibly alter the natural distribution of neutrophils. The visualization was accomplished using a Zeiss LSM-700 confocal microscope. (E) The graph presenting the influence of 100 nM PACAP-38 on the number of the neutrophils concentrated around right posterior lateral line neuromasts (PLL) (L1, LII.1, L2, LII.2, L3, L4, L5 and L6) after 10 µM CuSO₄ exposure. The presented values refer to the average number of neutrophils in each group. 100 nM PACAP-38 treatment resulted in a significant, over two-fold decrease in the number of the neutrophils found singly in the area defined by notochord borders and those associated with neuromasts as compared to that determined in the 10 µM CuSO₄-exposed group (one-way ANOVA, Kruskal–Wallis test with Dunn’s post-test, GraphPad Prism 5, <i>p</i> < 0.001). N/group = 15.</p

Morphology of L2 neuromast hair cells in 5 dpf Tg(pou4f3:GAP-GFP) zebrafish larvae.

<p>(A) control, (B) exposed to 10 µM CuSO4 for 40 min, and (C) exposed to the mixture of 10 µM CuSO₄ and 100 nM PACAP-38 for 40 min, following 1 hour pre-incubation with 100 nM PACAP-38 only (n/group = 15). The visualization was accomplished using a Zeiss LSM-700 confocal microscope. (A) Hair cells in the control group exhibited morphology without any necrosis features. (B) Copper exposure evoked severe necrosis, resulting in hair cell rosette disintegration. Hair cells were round-shaped and swollen (arrowheads). Other groups of hair cells appeared shrunken and fragmented (arrow), suggesting the involvement of other death pathways. (C) In the PACAP-38 co-treated group, the necrosis was comparably severe. The same necrotic signs, i.e. round-shaped and swollen (arrow heads), as well as shrunken and fragmented (arrow) cells were also observed.</p

A graph illustrating expression profiles of genes encoding three PACAP receptors (PAC1, VPAC1 and VPAC2) in samples from zebrafish 5 dpf larvae, kidney tissue and neutrophils.

<p>The material concerning larvae consisted of pooled individuals (n = 30), the kidney tissue refers to a single cell suspension derived from kidneys of five adult zebrafish and the neutrophil population consisting of 500,000 GFP+ sorted cells by FACS. Each collection was covered by samples analyzed in triplicate. Data in the figure represent the average of the representative experiment. Gene expression values were normalized to housekeeping gene β-actin. The existence of all types of PACAP receptor transcripts was reported in each collection, however at a different levels. In neutrophils, <i>adcyap1r1a</i> appeared as a predominant form for PAC1 and, for the VPAC family, the most prevalent was <i>vipr1b</i>. The lowest expression level was exhibited by <i>adcyap1r1b</i> and <i>vipr1a</i> genes. Interestingly, <i>adcyap1r1a</i> and <i>vipr1b</i> were expressed at similar levels in each studied sample, whereas for the remaining PACAP receptor genes, the highest expression levels were found in whole larvae, lower in the kidney tissue and the lowest was in neutrophils.</p

Expression profiles of pro-inflammatory and stress-inducible genes.

<p>The graphs present the mRNA expression of (A) interleukin 1β (<i>IL-1β</i>), (B) interleukin 6 (<i>IL-6</i>), (C) interleukin 8 (<i>IL-8</i>), (D) interleukin 10 (<i>IL-10</i>), (E) macrophage receptor MARCO (<i>MARCO</i>), (F) activating transcription factor 3 (<i>ATF3</i>), (G) tumor necrosis factor (<i>TNFα</i>), (H) C-X-C motif chemokine receptor 1 (<i>CXCR1</i>) and (I) C-X-C motif chemokine receptor 2 (<i>CXCR2</i>) from pooled (n = 30) 5dpf wild-type zebrafish larvae in four experimental groups: 1) control, 2) exposed to 10µM CuSO₄ for 40 min, 3) exposed to a mixture of 100 nM PACAP-38 + 10µM CuSO₄ for 40 min preceded by one hour pre-incubation with 100 nM PACAP-38 only, and 4) exposed to 100nM PACAP-38 only. Each group was covered by samples analyzed in triplicate in three separate experiments. Data in the figure represent the average of the three individual experiments. Gene expression values were normalized to housekeeping gene <i>β-actin</i>. To maintain image clarity, only differences between CuSO₄ exposed and PACAP-38 co-treated groups are marked on the graphs. Co-treatment with PACAP-38 significantly reduced up-regulated by copper treatment <i>IL-1β</i> and <i>IL-6</i>, <i>IL-8</i> and <i>ATF3</i> gene expressions (A, B, C and F) (one-way ANOVA, Kruskal–Wallis test with Dunn’s posttest, GraphPad Prism 5, <i>p</i> < 0.05), while it had no significant influence on <i>IL-10</i>, <i>MARCO</i> or <i>TNFα</i> genes (D, E and G) (one-way ANOVA, Kruskal–Wallis test with Dunn’s posttest, GraphPad Prism 5, <i>p</i> > 0.05). With regard to <i>CXCR1</i> and <i>CXCR2</i>, 10 µM copper chemical injury and 100 nM PACAP-38 treatment did not significantly change the expression level of the receptors. (one-way ANOVA, Kruskal–Wallis test with Dunn’s post-test, GraphPad Prism 5, <i>p</i> > 0.05).</p

Pituitary adenylate cyclase–activating polypeptide (PACAP-38) plays an inhibitory role against inflammation induced by chemical damage to zebrafish hair cells

<div><p>Pituitary adenylate cyclase–activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day post-fertilization (dpf) zebrafish larvae of three diverse genetic lines [transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen] were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO₄, (3) 10 µM CuSO₄+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO₄ solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated <i>in vivo</i> under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO₄-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO₄ exposure up-regulated the expression of <i>IL-8</i>, <i>IL-1β</i>, <i>IL-6</i> and <i>ATF3</i>, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. <i>Adcyap1r1a</i> and <i>vipr1b</i> appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.</p></div

A photograph illustrating the neutrophil counting area.

<p>5 dpf Tg(MPX:GFP) transgenic zebrafish larvae after 40 min exposure to 10 µM CuSO4 presents the area were neutrophils were quantified (green dots; in the intact larvae only single neutrophils were observed). Both neutrophils associated with investigated neuromasts (L1, LII.1, L2, LII.2, L3, L4, L5 and L6) as well as those which did not adhere directly to the neuromasts (but were sparsely found within the area encircled by the notochord [white arrow]) were counted. Dorsal and ventral myotomes marked with red arrows and terminal neuromasts (ter) were excluded from the analysis. The larvae carried myeloperoxidase promoter driving the expression of green fluorescent protein (GFP) in myeloid leukocytes (mostly neutrophils). The visualization was accomplished using a Zeiss LSM-700 confocal microscope.</p

Primers used in the study.

<p>Primers used in the study.</p