Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Evaluation of Leukocyte Mobilization and Platelet Aggregatory Effects of Ciprofloxacin, Lincomycin and Erythromycin in Wistar Albino Rats

This study evaluated leucocyte mobilization and platelet aggregation effects of ciprofloxacin, lincomycin and Erythromycin in Wistar albino rats. Thirty-three female Wistar albino rats weighing 130-160 g and three male Wistar albino rats weighing 188-194 g, fed commercial growers’ mash and clean tap water were used. In leukocytes assay, thirty-three adult female Wistar rats were assigned into five treatments, eleven groups of three rats per group. First three groups were treated 10 mg kg-1, 20 mg kg-1 and 40 mg kg-1 of ciprofloxacin, next three groups same doses of lincomycin, next three groups same doses of Erythromycin, the tenth group received Indomethacin 5 mg kg-1 (reference drug), the last group 5 mg kg-1 normal saline. Assay on platelet aggregator activity involved collection of blood samples (10 ml each, 1% EDTA, centrifuged at 3000 rpm for 10 minutes), test drugs dissolved in 1 mg/ml distilled water and Indomethacin (the reference drug). The three antibiotics significantly reduced leucocyte mobilization into the affected tissue. They all had their maximum inhibitory effects at the highest dose (40 mg kg-1) compared with indomethacin. Erythromycin 40 mg kg-1 showed the highest inhibitory effect on leucocyte migration. Whereas ciprofloxacin and erythromycin had stepwise increase in absorbance from time 0 secs through to time 120 secs, lincomycin showed a sharp decrease in the absorbance at around 30 seconds followed by a continuous increase up to 120 seconds. The testing drugs prevented leukocyte mobilization also had stepwise increase in absorbance from time 0 secs through to time 120 secs in platelet aggregatory activity assay to some extent.

Chemokines (CCL3, CCL4, and CCL5) Inhibit ATP-Induced Release of IL-1β by Monocytic Cells

Chemokines and ATP are among the mediators of inflammatory sites that can enter the circulation via damaged blood vessels. The main function of chemokines is leukocyte mobilization, and ATP typically triggers inflammasome assembly. IL-1β, a potent inflammasome-dependent cytokine of innate immunity, is essential for pathogen defense. However, excessive IL-1β may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1β. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with the P2X7 agonist BzATP for 30 min to induce IL-1β release. CCL3, CCL4, and CCL5 dose dependently inhibited BzATP-stimulated release of IL-1β, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. It was blunted after silencing CCR1 or calcium-independent phospholipase A2 (iPLA2) by siRNA and was sensitive to antagonists of nicotinic acetylcholine receptors containing subunits α7 and α9. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1β release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1β from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nicotinic acetylcholine receptor subunits α7 and α9. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1β is minimized.

Abstract 15294: Activation of Mac-1 Integrin Prevents G-CSF Mediated Monocyte Mobilization and Atherosclerosis Severity in Hypercholesterolemic Mice

Background: Leukadherins (LA) are a novel family of Mac-1 agonists that increase cell adhesion and prevent leukocyte mobilization and tissue inflammation. This study brings new insights into how leukadherin LA1 protects hypercholesterolemic ApoE-null mice from excessive atherosclerosis development. Hypothesis: Activation of Mac-1 integrin with leukadherin LA1 retains monocytes in medullary and extramedullary centers and therefore, controls high fat diet induced monocytosis and atherosclerosis in ApoE-null mice. Methods and Results: Once daily administration of LA1 (10mg/kg) for 16 weeks significantly reduced atherosclerosis in the entire aorta and the aortic valve of high fat diet fed ApoE-null mice as determined by Sudan IV staining. The LA1 treatment had not effect on body weight or plasma lipid levels though it significantly reduced the number of circulating monocytes (Lin2- CD11c- CD11b+ by FACS). The remaining circulating monocytes in LA1-treated mice displayed low levels of Ly6C, a marker for inflammation. Interestingly, LA1 caused monocyte retention in the bone marrow (BM) and macrophages (F4/80+ by IHC) in the spleen of hypercholesterolemic mice, which account for the low numbers of monocytes seem in the circulation of these mice. On the other hand, the excessive number of BM monocytes didn’t compromise the number of hematopoietic (Lin- Sca+ c-Kit+) or myeloid (Lin- Sca- c-Kit+) progenitor cells. Finally, we assessed the effect of LA1 on systemic inflammatory mediators using multiplex immunoassay. The plasma levels of G-CSF, one of the main monocyte mobilization cytokines capable of promoting atherosclerosis in ApoE-null mice, were found reduced in a half in treated versus control mice. Conclusions: These data demonstrate that Mac-1 activation with LA1 significantly reduces atherosclerosis in hypercholesterolemic ApoE-null by impairing G-CSF mediated monocyte mobilization from medullary and extramedullary centers.