For in vitro expression analysis
For in vitro expression analysis of CXCL8 and CXCRs by stimulated leucocytes, peripheral blood leucocytes (PBLs) were isolated using Percoll density gradient centrifugation as described earlier . Cell suspensions were adjusted to 5×10/ml. Four milliliters of tissue culture medium were plated per well of 6-well tissue culture dishes (Corning Incorporated, USA). The Imiquimod were then treated with 5μg/ml of PHA (Sigma), 5μg/ml of LPS ( 0111:B4, Sigma, USA), 100μg/ml of ConA (Wako) and 100μg/ml of Poly I:C (Amersham) (for doses, see Ref. ), and cultured in a humidified chamber with 5% CO at 20°C. Non-treated cells were set aside for controls. Cells from all treated groups were collected at 3, 6 and 12h. Total RNAs were isolated from all groups and first-strand cDNAs were synthesized as described earlier. The mRNA levels of the three genes were expressed as the ratio to that of β-actin, which was amplified in parallel to control for variation in mRNA concentration in the RT reaction. The optimal cycling profile for β-actin was 15s at 95°C, 15s at 50°C and 30s at 72°C for 30 cycles, using β-actin F/β-actin R primers (); that for CXCL and CXCRs was 15s at 95°C, 15s at 63°C and 30s at 72°C for 35 cycles, using CXCL8F1/CXCL8R2, QPA-F/CXCR1RA and QPB-F/CXCR2R2 primer pairs, respectively. The band intensities were quantified using software ‘QuantityOne’ (Bio-Rad). All the experiments were performed at least twice from eight individuals and each employed different batches of samples to validate the results. All data were expressed as the mean±one standard deviation (SD). The differences of means between control groups and treated groups were analyzed by the Student\'s -test. Results showed that LPS significantly downregulates the expression of CXCL8 by PBL in fugu (a). LPS also significantly downregulated the expression of CXCRs at all time points (b and c). In flounder, CXCL8 transcripts were detected in the leucocytes from the head kidney and spleen in the LPS-injected fish, while no transcripts were detected in unstimulated animals . Increased expression of CXCL8 was similarly observed in head kidney macrophages of rainbow trout in response to LPS and human TNF-α . In the common carp, in anterior kidney phagocyte-enriched leukocyte suspensions\' expression was upregulated by brief stimulation with PMA, but not LPS . These apparent differences might be attributable to different sources of the cells used for stimulation. Unlike all of those referenced fresh water species, fugu, which lives in salt water, encounters different kinds of challenges. But we are not sure whether this is the reason why fugu show a different kind of response to LPS. Moreover, fugu might have a different response to challenge by LPS as fugu seems to have no TLR4, which recognizes LPS in mammals. PHA upregulates CXCL8 and CXCRs in PBL even after only a short time period (). On the other hand, Poly I:C increased both CXCL8 and CXCR1 expression (a and 6b, respectively), while suppressing CXCR2 expression (c). Conversely, ConA was found not to significantly affect regulation of CXCL8 (a) or its receptors (b and 6c). The expression of CXCL8 and CXCRs in various tissues and their up- or down-regulation by different stimulators indicate that these genes are certainly involved in the immune system of fugu. Kuroda et al. reported that in fish ELR chemokines were angiogenic chemoattractants for neutrophils while non-ELR chemokines were angiostatic chemoattractants for lymphocytes . Until now, it has not been clear as to the immunological role played by CXCL8, which lacks ELR motif in teleosts. However, in fugu, CXCL8 as well as CXCRs was strongly expressed even in unstimulated tissues, though we cannot rule out the possibility that, unbeknownst to us, this specimen encountered antigenic challenges.
Introduction Angiogenesis, the process of endothelial sprouting then inducing the formation of new blood vessels, which is important for pregnancy, wound healing. Nevertheless, angiogenesis is essential for tumour growth and metastasis . Using anti-angiogenesis targeting drugs such as humanized antibody Avastin, tyrosine kinase inhibitor sorafenib, sunitnib has great potential in cancer therapy , however, the use of inhibitors of one growth factor or siganling intermediate only partly work in clinic. Worse, these drugs always enhance tumour hypoxia and then induce poor prognosis .