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Front Immunol
2018 Sep 12;9:2058. doi: 10.3389/fimmu.2018.02058.
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Amphibian (Xenopus laevis) Interleukin-8 (CXCL8): A Perspective on the Evolutionary Divergence of Granulocyte Chemotaxis.
Koubourli DV
,
Yaparla A
,
Popovic M
,
Grayfer L
.
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The glutamic acid-leucine-arginine (ELR) motif is a hallmark feature shared by mammalian inflammatory CXC chemokines such the granulocyte chemo-attractant CXCL8 (interleukin-8, IL-8). By contrast, most teleost fish inflammatory chemokines lack this motif. Interestingly, the amphibian Xenopus laevis encodes multiple isoforms of CXCL8, one of which (CXCL8a) possesses an ELR motif, while another (CXCL8b) does not. These CXCL8 isoforms exhibit distinct expression patterns during frog development and following immune challenge of animals and primary myeloid cultures. To define potential functional differences between these X. laevis CXCL8 chemokines, we produced them in recombinant form (rCXCL8a and rCXCL8b) and performed dose-response chemotaxis assays. Our results indicate that compared to rCXCL8b, rCXCL8a is a significantly more potent chemo-attractant of in vivo-derived tadpole granulocytes and of in vitro-differentiated frog bone marrow granulocytes. The mammalian CXCL8 mediates its effects through two distinct chemokine receptors, CXCR1 and CXCR2 and our pharmacological inhibition of these receptors in frog granulocytes indicates that the X. laevis CXCL8a and CXCL8b both chemoattract tadpole and adult frog granulocytes by engaging CXCR1 and CXCR2. To delineate which frog cells are recruited by CXCL8a and CXCL8b in vivo, we injected tadpoles and adult frogs intraperitoneally with rCXCL8a or rCXCL8b and recovered the accumulated cells by lavage. Our transcriptional and cytological analyses of these tadpole and adult frog peritoneal exudates indicate that they are comprised predominantly of granulocytes. Interestingly, the granulocytes recruited into the tadpole, but not adult frog peritonea by rCXCL8b, express significantly greater levels of several pan immunosuppressive genes.
Figure 1. In silico analyses of CXCL8 phylogeny and protein sequence conservation.(A) The protein alignment was performed using ClustalW2 server. Fully conserved residues are indicated by an asterisk (*), partially conserved and semi-conserved substitutions are represented by “:” and “.”, respectively. Putative signal peptides are bolded, the ELR motif is boxed in gray and the conserved CXC motif is highlighted. (B) The phylogenetic tree was constructed using the neighbor joining method and bootstrapped 10,000 times (denoted as %s). The accession numbers for the respective protein sequences are: guinea pig CXCL8: NP_001166870.1; naked mole rat CXCL8: XP_004833980; rhesus monkey CXCL8: NP_001028137.1; chimpanzee CXCL8: NP_000575.1; human CXCL8: NP_000575.1; horse CXCL8: NP_001077420.2; hare CXCL8: ALG04568.1; pig CXCL8: NP_999032.1; cow CXCL8: NP_776350.1; zebrafish CXCL8_L2: XP_009305130.1; carp CXCL8_L2: XP_018936341.1; chicken CXCL8: NP_990349.1; painted turtle CXCL8: XP_005304195.1; soft shelled turtle CXCL8: ACP28489.1; alligator CXCL8: XP_006018817.1; kiwi CXCL8: XP_013807042.1; barn owl CXCL8: XP_009963343.1; chimney swift CXCL8: XP_010001929.1; Himalayan frog CXCL8: XP_018421489.1; Western clawed frog CXCL8a: XP_002942578.2; African clawed frog CXCL8a: OCU00045.1; Western clawed frog CXCL8b: XP_002942578.2; African clawed frog CXCL8b: NP_0010912223.1; zebrafish CXCL8_L1: XP_001342606.2; carp CXCL8_L1: XP_016375461; trout CXCL8: XP_020330727.1; salmon CXCL8: NP_001134182.1; haddock CXCL8: CAD97422.2; gilthead seabream CXCL8: AGS55343.1; fugu CXCL8: NP_001027759.1; snapper CXCL8: AGV99968.1; banded houndshark CXCL8: BAB79448.1; whale shark CXCL8: XP_020370926.1; elephant shark CXCL8: NP_001279539.1.
Figure 2. Analyses of Cxcl8a and Cxcl8b gene expression in healthy and immune challenged animals. (A) Cxcl8a and Cxcl8b gene expression in tadpole and adult kidney (kid), liver (liv), muscle (mus), spleen (spl), skin (sk), bone marrow (bm), and intestine (int) tissues (N = 6). (B) Cxcl8a, Cxcl8b, and Gcsfr gene expression in tadpole and adult kidneys 3 days post FV3 infection (104 PFU/tadpole; 5 × 106 PFU/adult) (N = 5). (C) Cxcl8a and Cxcl8b gene expression in amputated tadpole tails (N = 5). (D) Cxcl8a and Cxcl8b gene expression in adult frog hind leg skin wounds (N = 5). All gene expression was compared relative to Gapdh control and all results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
Figure 3. Analyses of CXCL8a and CXCL8b gene expression in frog macrophages and granulocytes. (A) Cytology of tadpole rG-CSF-elicited peritoneal granulocytes (G-CSF-Grn) and adult frog bone marrow-derived macrophages, differentiated with rM-CSF (M-CSF-Mϕ) or rIL-34-Mϕs (L-34-Mϕ) and granulocytes (Grn), differentiated with rG-CSF (G-CSF-Grn). (B) The adult frog M-CSF-Mϕ, IL-34-Mϕ, and G-CSF-Grn were examined for their steady state gene expression of Cxcl8a and Cxcl8b. (C) The adult frog M-CSF-Mϕ, IL-34-Mϕ, and G-CSF-Grn were either mock infected (saline) or challenged with FV3 at a multiplicity of infection of 0.5 PFU/cell. After 24 h of challenge, cells were examined for Cxcl8a and Cxcl8b gene expression relative to Gapdh (N = 5). (D) M-CSF-Mϕ, IL-34-Mϕ, and G-CSF-Gran were mock challenged (saline) or challenged with heat-killed E. coli for 24 h before Cxcl8a and Cxcl8b gene expression analysis, relative to Gapdh (N = 5). All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
Figure 4. The rCXCL8a and rCXCL8b elicit distinct chemotaxis responses in tadpole and adult granulocytes. (A,B) Medium or increasing concentrations of rCXCL8a or rCXCL8b were loaded into bottom chemotaxis chamber wells and (A) tadpole or (B) adult frog granulocytes (105 cells/well) were loaded into top wells, separated by 5 μm pore filters. After 3 h of incubation, the filters were stained with Giemsa, mounted bottom side up and the numbers of migrating cells per field of view enumerated. Cells from three individual animals were used for each chemokine concentration (N = 3) and the highest tadpole (10−4 ng/ml of rCXCL8a; 101 ng/ml of rCXCL8b) and adult frog (10−6 ng/ml of rCXCL8a; 10−4 ng/ml of rCXCL8b) granulocyte chemotactic activities were confirmed in two additional, independent experiments and the results combined and presented here (N = 9). Chemokinesis was measured by adding respective concentrations of rCXCL8 or rCXCL8b that elicited maximal cell migration, to both lower and upper chambers. (C) Tadpole and adult frog gene expression of Cxcr1 and Cxcr2 relative to Gapdh. All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
Figure 5. Roles of the frog CXCR1 and CXCR2 in the rCXCL8a- and rCXCL8b-elicited chemotaxis. (A) Tadpole and (B) adult frog granulocytes (105 cells/well) were subjected to chemotaxis assays in absence or presence of 1 nM or 100 nM of the CXCR1/CXCR2 inhibitor, reparixin and using the respectively most chemo-attractive concentrations of rCXCL8 or rCXCL8b. Cells from three individual animals were used for each chemokine concentration (N = 3). (C) Tadpole and (D) adult frog granulocytes (105 cells/well) were subjected to chemotaxis assays in absence or presence of 5 or 100 nM of the CXCR2 inhibitor, SB265610 and using the respectively most chemo-attractive doses of rCXCL8 or rCXCL8b. Cells from four individual animals were used for each chemokine concentration (N = 4). All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
Figure 6. Analysis of immune gene expression and cytology of rCXCL8a- and rCXCL8b-elicited tadpole cells. Tadpoles and adult frogs were injected intraperitoneally with rCXCL8a or rCXCL8b (1 μg/g of body weight) in 10 μl of saline or with an equal volume of the vector control (supernatants from empty vector-transfected Sf9II cell, processed in parallel to rCXCL8a and rCXCL8b production). After 4 h, animals were lavaged with saline and the cells were enumerated (A). The tadpole (B,C) and adult frog (not presented) rCXCL8a and rCXCL8b-elicited cell immune gene expression was examined relative to the Gapdh control (N = 6). All results are presented as means + SEM. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not. (D) Tadpole rCXCL8a and rCXCL8b-elicited cells were stained with a rabbit anti-frog rG-CSFR (or saline) Ab and secondary goat anti-rabbit Ab and examined by confocal microscopy.
Supplemental Figure 1. Western blot of the produced, purified, and eluted rCXCL8a and rCXCL8b fractions. The eluted recombinant (r)CXCL8a and rCXCL8b were resolved by SDS PAGE, transferred onto nitrocellulose membranes and western blots were performed using an HRP-conjugated mouse anti-V5 (Sigma) to determine which elution fractions contained rCXCL8a (15 kDa) and rCXCL8b (16 kDa).
Supplemental Figure 2. Analyses of the anti-G-CSFR polyclonal antibody and the rCXCL8a- and rCXCL8b-elicited granulocyte expression of the Cxcr1 and Cxcr2 genes. (A) Rabbits were immunized with rG-CSFR and the resulting serum was applied to a HiTrap Proetin A HP column (GE Health) to isolate the IgG fraction, and to a rG-CSFR-bound Sulfo-Link Protein column to purify the IgG fraction that cross-reacted with the rG-CSFR. To confirm the specificity of this reagent, this anti-rG-CSFR IgG farction was used to perform a western blot of the rG-CSFR before (lane 1) or after (lane 2) pre-absorbing the Ab against the rG-CSFR. (B) Tadpoles and adult frogs were injected intraperitoneally with rCXCL8a or rCXCL8b (1 μg/g of body weight) in 10 μl of saline or with an equal volume of the vector control. After 4 h, animals were lavaged with saline and the cells were enumerated and examined for their expression of Cxcr1 and Cxcr2. Above-head letters denote statistical designations: experimental groups described by distinct letters are statistically different (P < 0.05) while those marked by the same letters are not.
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