Riboflavin as a Source of Autofluorescence in Eisenia fetida Coelomocytes

Photochemistry and Photobiology, Mar/Apr 2006 by Koziol, Beata, Markowicz, Magdalena, Kruk, Jerzy, Plytycz, Barbara

ABSTRACT

Immunocompetent cells of earthworms (coelomocytes) contain adherent amoebocytes and large eleocytes (chloragocytes); the latter are filled with numerous granules. We have previously shown that eleocytes of several (but not all) earthworm species exhibit strong autofluorescence detectable by fluorescent microscopy and flow cytometry. In the present article, the molecular origin of eleocytes autofluorescence was elucidated in coelomocytes expelled via dorsal pores in the integument of Eisenia fetida subjected to electric shock (1 min at 4.5 V). Spectrofluorometry (excitation and emission spectra and fluorescence lifetime), together with HPLC analysis of coelomocyte suspensions and supernatants, indicated that riboflavin but not FMN (flavin mononucleotide) or FAD (flavin-adenine dinucleotide) is the main fluorophore responsible for eleocyte fluorescence in this species. Additionally, lipofuscins are suspected to participate in this phenomenon.

INTRODUCTION

The coelomic cavity of earthworms may be inhabited by various sorts of soil-derived parasites, such as bacteria, gregarines and fungi, which are kept in check by the combined activities of the earthworm immunocompetent cells (coelomocytes) and humoral factors (1,2). Coelomocytes are involved in phagocytosis (3), cytotoxicity (4-6) and encapsulation of invading parasites within so-called brown bodies (7). They also participate in humoral immunity through their secretions (e.g. lysozyme [8] and fetidins [9]).

Coelomocytes have been classified largely on the basis of differential staining (10), ultrastructural morphology, the chemical nature of their inclusion granules and functional traits, such as adherence and chemotaxis (11). Two main populations of coelomocytes-amoebocytes and eleocytes-are present in various proportions in different species (12,13). The origin of these cell types is still a matter of controversy. There are indications that amoebocytes derive from the mesenchymal lining of the coelom (11), whereas eleocytes (chloragocytes) differentiate from the chloragogen cells that cover the coelomic surfaces of the alimentary tract and major blood vessels (14). Eleocytes participate in the metabolism and storage of glycogen and lipids and they transport nutrients systemically by means of coelomic fluid to diverse cells and tissues (14).

Analysis by phase-contrast fluorescent microscopy and flow cytometry demonstrated that eleocytes of some earthworm species exhibit a strong autofluorescence (15). A high percentage (>10%) of autofluorescent eleocytes was observed in 6 of 12 investigated earthworm species: Dendrobaena veneta (2), Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida and Octolasion organisms (O. cyaneum, O. tyrtaeum tyrtaeum and O. tyrtaeum lacteum). In contrast, autofluorescence was observed in

MATERIALS AND METHODS

Animals. Adult (i.e. sexually mature, with a well-developed clitellum) E. fetida (Savigny 1826), were bought in 2002 from the commercial supplier Vitahum (Nowy Sacz, Poland) and then bred at room temperature at the Institute of Zoology, Jagiellonian University (Krakow, Poland), in plastic-boxes filled with soil (16,17). The earthworms were fed Hour twice per week.

Harvesting of coelomocytes. Earthworms were stimulated for 1 min with a 4.5 V electric current to expel coelomic fluid containing coelomocytes through the dorsal pores in the integument, according to a modified version of the procedure described by Roch (18). Briefly, after weighing, washing and dry blotting, single earthworms were placed in separate Petri dishes containing 3 mL of extrusion fluid (PBS supplemented with 2.5 g/L EDTA [Sigma]) to prevent cell aggregation (12,13). Extruded coelomocytes were counted in a haemocytometer and adjusted to a concentration of 10^sup 6^ cells/ mL. Cell viability was assessed using a Try pan Blue exclusion test (19), after mixing of equal volumes of coelomocyte suspensions and 0.4% Trypan Blue (Sigma) solution. Cell viability was always >95%. Cells were imaged by fluorescent microscopy (Jenamed 2, Carl Zeiss Jena; objective 25�) and photographed (Coolpix 4500 digital camera). Each animal was used only once and coelomocytes from each particular earthworm were analyzed individually.

Flow cytometry. Fresh coelomocytes were analyzed with a FACScalibur flow cytometer (BD Biosciences). During analytical experiments 10 000 events per worm sample were collected and forward and side scatter and FL-1H autofluorescence were recorded. The resulting FCS files were analyzed using WinMDI 2.8 software (kindly provided by Joe Trotter; available at: http://facs.scripps.edu) on dot plots of cell complexity/granularity (side scatter) versus FL1-H autofluorescence, permitting identification of cells that were strongly autofluorescent.

Spectrofluorometric measurements. The spectrofluorometric measurements were performed on coelomocyte suspensions, as well as on the cell supernatants after centrifugation of the suspensions on a benchtop Eppendorf centrifuge at 12 000 r.p.m. for 10 min. The fluorescence measurements were performed with a Perkin-Elmer spectrofluorometer LS50B (Beaconsfield, Buckinghamshire, United Kingdom) in 1 � 1 cm cuvettes with an excitation slit of 5 nm and an emission slit of 5 or 10 nm. Excitation spectra were recorded between 300 and 520 nm with emission at 525 nm, whereas emission spectra were recorded between 400 and 700 nm with excitation at 370 nm. Fluorescence-lifetime measurements were performed using Phase Modulation Fluorometer ISS K2 (Urbana, IL) with the following parameters: excitation at 370 nm, emission-cutoff filters at 370 nm and 450 nm and 12 modulation frequencies from 2-250 MHz.

HPLC. The coelomocyte supernatants obtained as described above were mixed with methanol in 60:40 (vol/vol) proportion and analyzed by HPLC with a C18 reverse-phase column in water:methanol (60:40 [vol/vol]) at a flow rate of 0.8 mL/min. Fluorescence detection was performed with excitation at 370 nm and emission at 530 nm. Flavin-adenine dinucleotide (FAD) was from Sigma, flavin mononucleotide (FMN) from Serva and riboflavin from Reanal (Budapest, Hungary).