Phagocytosis and Respiratory Burst Activity in Lumpsucker (Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes

Isolated leucocytes show strong respiratory burst activity upon stimulation with PMA.

<p>Flow cytometry of respiratory burst in leucocytes from PBL (A), HKL (B) and SL (C). FL1 (green fluorescence) histograms show RHO fluorescence after PMA stimulation. Stimulated cells: 0.1 µg ml⁻¹ of PMA, red line and 1 µg ml⁻¹ of PMA, blue line. Controls: Non-stimulated cells without PMA and with DHR (grey filled peak), used for determination of limit between RHO positive and negative cells. Positive control for oxidation of DHR by H₂O₂ to RHO (grey line). Other negative controls: without PMA and DHR (aqua line), with PMA and without DHR (green line) and with H₂O₂, but without PMA and DHR (yellow line). These negative controls without DHR have low fluorescent intensity. Horisontal bars indicate RHO positive cells. The corresponding size/granularity (FCS/SSC) dot plots of PBL, HKL and SL show RHO positive cells (red) for PMA (1 µg ml⁻¹) stimulated cells.</p

Lumpsucker kidney used for isolation of leucocytes.

<p>Dissected kidney where the section used for isolation of leucocytes from the left cranial lobe is marked.</p

The phagocytic capacity of isolated leucocytes is high.

<p>FL1 (green bead fluorescence) histograms (left) showing phagocytic capacity of PBL (A), HKL (B) and SL (C) incubated with fluorescent beads (1 µm) for 4 h. Increased peak fluorescence indicates an increased number of ingested beads. Picture insets show cells stained with Colorrapid from PBL, HKL and SL samples that have ingested various numbers of beads. The left dot plots show cells in the red (cells with 1 bead) blue (cells with two beads) and green (cells with 3 or more beads) and black (non-phagocytic cells) regions; cells with a higher number of ingested beads have a higher granularity (SSC-value). The dot plots to the right show the light scatter properties of the cells incubated without beads at the instrument settings used for the phagocytosis assay.</p

The proportions of RHO-positive cells and geometric mean fluorescence intensity (GMFI) of PMA stimulated PBL, HKL and SL from lumpsucker analysed by flow cytometry.

a<p>N = 4, ^bN = 5</p

Flow cytometry analyses of leucocytes isolated from peripheral blood, head kidney and spleen.

<p>Representative size/granularity (FSC/SSC) dot plots, show different sub populations among PBL, HKL and SL. The regions used in the analyses, representing the live cells, are delimited in each panel.</p

PBL samples from fish with purple/red serum give yellow-orange autofluorescence in flow analyses.

<p>The lumpsuckers varied in color from green to brown and red (A). Samples of serum from different fish (B). Histograms and dot plots (insets) when PI is added to leucocytes (C). In histogram: Gray without PI and black with PI. Horisontal bars show PI positive cells. This sample is used for gating of live cells to exclude dead cells from the analyses. The left figure in C shows PI positive cells (red dots) in dot plot for PBL from the green lumpsucker giving serum no 3, while the right figure shows the same results for the brown/red fish giving serum no 7. Note the difference in the yellow-orange fluorescence (red dots in C) for these two PBL samples.</p

Morphological and cytochemical analyses of leucocytes isolated from peripheral blood, head kidney and spleen.

<p>Cytospin preparations of PBL, HKL, SL stained with Colorrapid (CR) (A), PAS (B) and MPO stained cells (C). The overview photos in A (left) and representative single cells (right), captured 630× magnifications. <i>i</i> = lymphocytes, <i>ii</i> = monocytes/macrophages, <i>iii</i> = polymorphonucear cells and <i>iv</i> = dendritic-like cells. The inset at top (right) in (A) show a polymorphonucleated cell (neutrophil) isolated from Atlantic salmon for comparison. In (B) and (C), representative single cells of isolated PBL, HKL and SL shown are captured at 630×. Representative cells from human blood smears are shown as controls (630×). Note that PAS staining (B) was highly variable and the two negative cells shown for PBL and HKL might be considered weak positive. In (C), overview of MPO stained SL and PBL, captured at 400×, show positive and negative leucocytes. Erythrocytes stain MPO positive. Scale bars = 5 µm.</p

Isolated leucocytes have potent phagocytic ability.

<p>Proportions of phagocytic leucocytes of total PBL, HKL and SL after 1, 4 and 8 hours ingestion of fluorescent beads measured by flow cytometry (mean, bars indicate SD, N = 6).</p

Phagocytic cells ingest beads rapidly.

<p>Proportions of phagocytic cells with various numbers of ingested beads in PBL, HKL and SL after incubation with fluorescent beads (1 µm) for 1, 4 or 8 h detected by flow cytometry (mean, bars indicate SD, N = 6 in all analysis except for SL 4 h where N = 5).</p