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Department of Biology, Hunter College, New York, New York 10021
The granular amebocyte is the single cell type in the general circulation of the horseshoe crab, Limulus polyphemus, and functions as the most important element in the immune system of the animal. The cytoplasm of the cell is packed with granules containing multiple immune effector proteins and peptides (1). Degranulation of the amebocyte, with the concomitant release of this complex of antimicrobial effectors, can be elicited by specific secretagogues such as bacterial lipopolysaccharide (LPS) (2). LPS is an essential component of the cell wall of all gram-negative bacteria and is an indicator molecule for the presence of these bacteria. Although LPS had previously been thought to be unique to gram-negative bacteria, a similar molecule has recently been found in the eukaryotic green algae, Chlorella, strain NC64A, maintained in bacteria-free culture (3). Algal LPS, like bacterial LPS, is composed of lipid A, 
-myristic acid, and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO). This material gelates the Limulus amebocyte lysate, a standard test for LPS (3). However, its biological activities are essentially uncharacterized.
Does algal LPS operate as an agonist of exocytosis of the granular amebocyte? Algal LPS was prepared as described previously (3). To evaluate the ability of the granular amebocyte to react to algal LPS, cultured horseshoe crab blood cells were challenged with algal or bacterial LPS, and the extent of exocytosis was evaluated by microscopic inspection (2). The animal was chilled for 2 h at 4 °C and bled (cardiac puncture with a 20-gauge needle) directly onto microscope coverslips submerged in cold LPS-free 3% NaCl. The cells were allowed to attach to the coverslips for 5 min; then the coverslips were assembled into perfusion chambers (with the cell-coated slips supported above glass slides with chips of #1
coverslips) as described by Armstrong and Rickles (2). The chambers were perfused with bacterial or algal LPS in 3% NaCl + 10 mM CaCl2, at room temperature, and observed over time either with a Nikon inverted phase contrast microscope, or a Zeiss phase contrast microscope equipped with a Nikon Coolpix digital camera. Control cultures were perfused with LPS-free 3% NaCl + 10 mM CaCl2. All glassware used was rendered LPS-free by heating at 180–200 °C for at least 4 h. Alternately, blood was collected directly into prechilled, virgin 35 x 10 mm plastic petri dishes containing 2 ml of LPS-free 3% NaCl, 1 drop of blood per dish. After allowing 5 min for the cells to attach to the dish, the culture was renewed with 2 ml fresh 3% NaCl. Blood plasma accelerates the exocytosis response in the absence of LPS (4), and its removal stabilizes the cells from spontaneous degranulation. Three percent NaCl was replaced with test solutions containing LPS in 3% NaCl + 10 mM CaCl2. Ca++ is required for cell flattening and a robust reaction to endotoxin. As before, the extent of exocytosis was determined by microscopic inspection.
The granular amebocyte responds positively to algal LPS (degranulation occurs), but the cell requires a concentration about 10 times higher than for bacterial LPS (E. coli Serotype 0127:B8, Sigma Cat # L-3129). Bacterial LPS provokes a vigorous exocytosis response at 5 µg/ml, causing the majority of the cells to degranulate within 20 min of exposure, whereas the cultured blood cell requires 50 µg/ml of algal LPS to initiate a similar response within that period (Fig. 1).
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The response of the granular amebocyte to algal LPS is interesting, both for the characterization of a LPS-like molecule from algae, and for a better understanding of the immune system of the animal. In this latter regard, algal colonization and subsequent erosion of the carapace of the horseshoe crab appears to be an important cause of mortality of the adult animal (7), so it is of interest to characterize the different ways by which the immune system can interact with algae. Our documentation of the parallel activities of algal and bacterial LPS in the induction of the exocytotic response of the granular amebocyte indicates that both molecules are capable of activating this important pathway of immunity in Limulus. This is the first demonstration that the LPS-like agent from an alga mediates LPS-like biological activities. We thank Mr. Jim Barkes of Nikon for assistance with microscopy and Dr. Norman Wainwright and Ms. Alice Childs for conducting LAL assays of algal LPS. This research was supported by NSF Grant MB 26771 (to PBA) and a fellowship from the Hunter College-Howard Hughes Medical Institute Undergraduate Education Program (to MLC).
Footnotes
1 School of Biological Sciences, University of Nebraska, Lincoln, NE 68583. 
2 Department of Molecular and Cellular Biology, University of California, Davis, CA 95616
Literature Cited
This article has been cited by other articles:
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  K-G. Lee, A. Braun, I. Chaikhoutdinov, J. DeNobile, M. Conrad, and W. Cohen Rapid Visualization of Microtubules in Blood Cells and Other Cell Types in Marine Model Organisms Biol. Bull., October 1, 2002; 203(2): 204 - 206. [Full Text] [PDF]
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