Identification of histones as endogenous antibiotics in fish and quantification in rainbow trout (Oncorhynchus mykiss) skin and gill.

Antimicrobial polypeptides (AMPPs) are increasingly recognized as a critical component of innate host defense. Among the AMPPs, polypeptides related to histones have been identified from many animals. Using peptide mapping, we further confirm the identity of two histone-like proteins from fish as members of the H2B (sunshine bass) and H1 (rainbow trout) histone groups. We optimized the conditions for measuring rainbow trout HLP-1/H2B via sandwich ELISA. We used two antibodies, one to the amino terminus and one to the carboxyl terminus, of trout histone H2B, as the capture antibodies, and we used peroxidase-labeled antibody raised to calf histone H2B as the secondary antibody. Specificity of the detecting antibody was confirmed by specific reactivity with histone H2B in tissue extracts via western blotting. The test was reproducible and capable of detecting as little as 5 ng of histone H2B (0.05 µg/ml). Histone H2B levels expressed in gill tissue of juvenile, healthy rainbow trout were well within concentrations that are lethal to important fish pathogens. However, there was a significant, age (size)-dependent decline in histone H2B concentrations as fish matured, until levels became virtually undetectable in market-size fish. In contrast, levels in skin appeared to remain high and unchanged in small versus large fish. Antibacterial activity in skin and gill tissues was closely correlated with histone H2B concentration measured via ELISA, which supports our previous finding that histones are the major AMPPs in rainbow trout skin and gill.

A novel mechanism of immune regulation: interferon-gamma regulates retention of CD4 T cells during delayed type hypersensitivity.

The local immune response is characterized by an increase in the rate of entry of lymphocytes from the blood into regional lymph nodes and changes in the output of cells in lymph. While significant data are available regarding the role of inflammation-induced vascular adhesion processes in regulating lymphocyte entry into inflamed tissues and lymph nodes, relatively little is known about the molecular processes governing lymphocyte exit into efferent lymph. We have defined a novel role for lymphatic endothelial cells in the regulation of lymphocyte exit during a delayed type hypersensitivity (DTH) response to mycobacterial purified protein derivative (PPD). Soluble, pro-adhesive factors were identified in efferent lymph concomitant with reduced lymphocyte output in lymph, which significantly increased lymphocyte binding to lymphatic endothelial cells. While all lymphocyte subsets were retained, CD4+ T cells appeared less susceptible than others. Among a panel of cytokines in inflammatory lymph plasma, interferon (IFN)-gamma alone appeared responsible for this retention. In vitro adhesion assays using physiological levels of IFN-gamma confirmed the interaction between recirculating lymphocytes and lymphatic endothelium. These data demonstrate a new level of immune regulation, whereby the exit of recirculating lymphocytes from lymph nodes is selectively and sequentially regulated by cytokines in a manner equally as complex as lymphocyte recruitment.

Pulmonary surfactant proteins A and D directly suppress CD3+/CD4+ cell function: evidence for two shared mechanisms.

Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3(+)/CD4(+) lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-gamma when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca(2+) ](i)), but not thapsigargin-induced changes in [Ca(2+)](i). In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca(2+)](i).