LPS-induced stimulation of phagocytosis in the sipunculan worm Themiste petricola: Possible involvement of human CD14, CD11B and CD11C cross-reactive molecules

Developmental & Comparative Immunology, Vol. 21, No. 4, pp. 349-362, 1997 0 1997 Elsevier Science Ltd. All rights reserved

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LPS-INDUCED STIMULATION OF PHAGOCYTOSIS IN THE SIPUNCULAN WORM Themiste petricola: POSSIBLE

INVOLVEMENT OF HUMAN CD14, CDllB AND CDllC CROSS- REACTIVE MOLECULES

Guillermo A. C. Bfanco,* Ana M. Escalada, Elida Alvarez* and Silvia Hajos*

IDEHU lnstituto de Estudios de lnmunidad Humoral UBA-CONICET, Chtedra de lnmunologia Facultad de

Farmacia y Bioquimica-Universidad de Buenos Aires, Buenos Aires, CP 1113, Argentina

(Received July 1996; Accepted January 1997)

OAbstract-Coelomocytes of Themistepetri- cola, a marine invertebrate of the phylum Si- puncula, were exposed in vitro to bacterial lipopolysaccharides (LPS), and the phagocytic activity against heat-killed yeast (Sacchar- omices cerevisiue) was evaluated using a flow cytometric assay. An increase of phagocytic activity was observed following pre-incubation of coelomocytes over 20 h with either 5 ug/mL LPS or 1.5 ug/mL phorbol12-myris- tate 13-acetate (PMA). The phagocytic enhancement induced by LPS was blocked by co-incubation with polymixin B, a ligand for the lipid A region of LPS. In a 72 h stimulation assay, LPS was also found to enhance phagocytosis. The enhancement was signifi- cantly higher when coelomocytes were incubated with LPS plus coelomic plasma. Using mAbs directed against human CD14 and components of the human LFA-1 complex, we identified coelomocyte surface antigens cross-reactive with CD14, CDllb and CDllc. The expression of CDllb and CDllc antigens was augmented by LPS treatment of coelomocytes. By double Buorescence assays, using mAb Leu-M3 and fluorescein labeled yeast, phagocytic coelomocytes were found to be mainly anti-CD14 positive. No cross-reac- tions were detected with mAbs against

Address correspondence to Dr Guillermo Blanco, Catedra de Inmunologia, Facultad de Farmacia y Bioquimica-UBA, Junin 956 4” Piso, Buenos Aires-CP 1113, Argentina. *Members of the research career (CONICET)

CDlla and CD18. Enzymatic treatment of coelomocytes with phosphatidyl inositol phospholipase C (PI-PLC) reduced the expression of the CD14-like antigen. The presence, in sipunculan coelomocytes, of antigens cross-reactive with CD14, the u chain of CR3 and of p150,95 raises the possi- bility that molecules related, although not necessary homologous, to the mammalian counterparts may have a role in the defense systems of these animals. 0 1997 Elsevier Science Ltd.

q Keywords-Z’Themistepetricolu; Sipuncula; Phagocytosis; LPS; CD14; CR3; p150,95.

Introduction

Invertebrate defense systems are characterized by the absence of lymphoid components. Nevertheless, such systems have cellular and humoral components that are also present in vertebrate immune systems: phagocytosis, cytotoxicity, complement-like system, cytokines, and various humoral components which seem to be relatively specific to invertebrate animals (l-3). Phagocytosis has been the effector function most commonly studied in these animals and the interaction with serum opsonins has been a matter of research interest in the last decade (l-4).

Bacterial lipopolysaccharides (LPS) are

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well known for their actions on mammalian cells with a direct or indirect role in immune responses (56). LPS have a definite role in the acute phase response affecting a multiple event process where cellular and humoral defense mechanisms are activated simultaneously or in se- quence (7,8). The presence of LPS in mammalian plasma activates the complement system, while polymorphonuclear cells (PMN) and monocytes are activated through receptors located at the plasma membrane (8,9). The cellular activation process includes events such as oxidative burst, cytokine production, activation of adhesion molecules and nitric oxide production (8,11-14). Some of these LPS-induced activation events are also observed in endothelial cells under appropriate conditions (8,15,16).

In PMN many of these responses to LPS appear dependent in part on enhanced function of the three members of the CD1 l/CD18 family of adhesion receptors: LFA- 1 (CD1 1 a/CD 1 S), CR3 (CDllb/CD18), and p150,95 (CDllc/ CD 18), also known as the pz or leukocyte integrins (17). Adhesion of PMN to endothelium requires the participation of CD 1 l/CD 18 molecules (18) and blockade of CD1 8 with mAbs prevents accumulation of PMN at the site of injury (19,20). Adhesion of PMN to protein-coated glass, enhanced oxidative burst in response to soluble agonists, and micro- bicidal activity are also dependent on increased CD 11 /CD 18 function (21,22). LPS has been shown to induce a dramatic enhancement of the adhesive function of CR3 after forming a complex with serum LPS binding protein (LBP) or septin (22,23).

Mononuclear cells respond to LPS by a mechanism where LPS first binds to the serum protein LBP and the resulting complex LPS-LBP is then recognized by CD14, a 55 kDa glycoprotein that is strongly expressed on monocytes and macrophages (24)(25). LBP and CD14 serve two physiological roles. These

proteins act as opsonin and opsonic receptor, respectively, to promote the phagocytic uptake of bacteria or LPS- coated particles by macrophages. They also enhance the ability of mononuclear cells to synthesize TNF-a in response to endotoxin (26). Addition of LBP accel- erates the synthesis of TNF-a and enables a response to doses of LPS lOO-fold lower than are otherwise required (21,27). Blockade of CD14 with mAbs prevents the synthesis of TNF-a by monocytes, in response to nanomolar concentrations of LPS in the presence of plasma (24,28).

Sipuncula is a phylum of non-segmen- ted coelomate marine worms. It has been phylogenetically related to Annelida although distinguished by the absence of segmentation in both developmental and adult stages (29). Sipuncula has also been considered close to Mollusca in phylo- geny, based on developmental evidence (30). Phagocytic coelomocytes play an essential role in the immune system of sipunculans as occurs in other invertebrates (31). In previous work we have shown that coelomocytes from Themiste petricola are capable of phagocytosing yeast under plasma-free conditions, although higher rates of phagocytosis were obtained when yeast cells were opsonized with coelomic plasma (31). In this study our main concern was to determine whether LPS and PMA were able to modulate the sipunculan phagocytic response in vitro. In addition, using mAbs directed against human molecules relevant to the acute phase response, we searched for cross-reactive antigens in sipunculan coelomocytes and explored the modulation of this antigens by LPS.

Our results showed that LPS specifi- cally increased the phagocytic activity of coelomocytes against HKSC. When coelomic plasma was present in the culture media the enhancement induced by LPS was higher. Labeling studies revealed the presence of CD14, CDllb and CDllc positive coelomocytes in Themiste petricola, and an increase in the proportion of

LPS-stimulation of phagocytosis 351

CD1 lb and CD1 lc positive cells after stimulation with LPS.

Materials and Methods

Animals

Adult Themiste petricola with an average wet weight of 100 mg were collected from crevices in intertidal rocks, at Santa Elena beach on the coast of Argentina, 34”s latitude, and main- tained in 500 mL plastic boxes with frequently renewed filtered sea water (3.2% salinity) at 18°C.

Culture Medium

Balanced salt solution (BSS), made to resemble the ionic composition of The- miste petricola coelomic fluid, was used for primary in vitro cultures of coelomocytes and for other experiments. The composition of BSS was: 0.4 it4 NaCl, 10.3 mM KCl, 10.1 mM CaCl*, 30.2 mM MgS04, 26.2 mM NaHCOs, pH 7.0. Overnight or 72 h cultures were held in RPM1 1640 supplemented with NaCl, KCl, CaC12, MgS04 and NaHCOs, as required to achieve the same concentrations cited for BSS. Coelomocyte viabi- lity, determined by Trypan Blue exclusion, remained as high as 99% for at least 9 h in BSS and more than 72 h in RPM1 1640 at 18°C.

Coelomocyte Isolation and Stimulation

Coelomocyte suspensions were pre- pared as indicated previously (31). In brief, coelomic fluid was harvested by incision of the body wall with a sterile surgical blade, allowing the fluid to drip into a 15 mL sterile centrifuge tube cooled to 4°C. Germinal cells were excluded with sterile nylon linen (mesh:

50 urn). Coelomocyte-free fluid (plasma) was separated after centrifugation and decanted into a sterile tube for further use in LPS stimulation experiments. The coelomocyte pellet was resuspended in BSS, washed three times by centrifugation in BSS and finally suspended in RPM1 1640. The corresponding stimulating agent was further added, and coelomocytes were cultured for 24,48 and 72 h in plastic tubes (Falcon, Lincoln Park, NJ).

Yeast Preparation

Heat-killed Saccharomyces cerevisiae (HKSC) was labeled with fluorescein isothiocyanate (FITC) (Sigma Chemical Co., St Louis, MI) according to the technique described by Pertircari et al. (32) with few modifications. One milliliter volume of 0.1 A4 carbonate buffer, pH 9.6 containing 10’ HKSC yeast cells and 0.3 ug/mL of FITC, was incubated at 4°C for 24 h. Fluoresceinated HKSC was washed three times in BSS and stored at - 20°C in small aliquots.

LPS, Polymixin B and PMA

Escherichia coli 011 l:B4 LPS and polymixin B 6000 USP units/mg (both from Sigma Chemical Co.) were diluted in sterile saline at 50x treatment concentrations. When polymixin B was used to block LPS effects, it was added immedi- ately prior to the LPS dose. LPS was tested at various concentrations in preliminary experiments and finally used at either 2 or 5 ug/mL. Phorbol 1Zmyristate 13-acetate (PMA) (Sigma Chemical Co.) was dissolved in DMS9 at a concentration of 0.75 mg/mL and stored at - 30°C.

Coelomocyte Stimulation and Phagocytosis Assay

Coelomocytes were incubated with


5 ug/mL LPS, either in the presence or absence of 10 ug/mL polymixin B, with 1.5 ug/mL PMA, or with 10 ug/mL polymixin B alone, in RPM1 1640 in sterile plastic tubes (Falcon, Lincoln Park, NJ) for 20 h at 18°C. Cells were then washed three times in BSS and 100 uL containing lo7 coelomocytes were placed in 96-well culture plates. One hundred microliters containing 1 O7 FITC-HKSC was added to each well and the mixture was incubated at 18°C for 45 min. Cells were then transferred to FACScan tubes, resuspended in 1 mL BSS and analyzed. Quenching of non- endocytosed HKSC was achieved with 20 uL of a 2 mg/mL suspension of Trypan Blue (32).

To evaluate further the role of plasma in the LPS induced stimulation, coelomocytes were cultured for 72 h under four different conditions: plasma-free culture medium, plasma-free culture medium containing 2 ug/mL LPS, medium containing 2 ug/mL LPS plus coelomic plasma diluted l/20, and medium containing plasma diluted l/20 alone. LPS was incubated in plasma for 15 min before being added to the culture media.

PI-PLC Treatment

Coelomocytes (1 x 106) were incubated for 1 h at 18°C in culture media containing 10 U/mL phosphatidyl inositol-speci- fit phospholipase C (PI-PLC) (Sigma Chemical Co., St Louis, MI). Controls were incubated in culture medium alone.

Monoclonal Antibodies and Coelomocyte Fluorescence Staining

Monoclonal antibodies used in this study were as follows: anti-CD 11 a (IgG2a; clone G25.2) phycoerythrin (PE) labeled 2.5 ug/mL in BSS; Leu-15 (CD 1 lb; IgG2a; clone D12) PE labeled 20 ug/mL; FHCL-3 (CDllc; IgG2b;

clone SHCL) PE labeled 2.5 ug/mL; Leu-MS (CD18; IgGl; clone L130) FITC labeled 20 ug/mL; Leu-M3 (CD14; IgG2b; clone Mo/P9) PE labeled 20 ug/ mL; mouse isotype controls (IgGl clone x40, IgG2a clone x 39, IgG2b clone x 75) PE or FITC labeled 20 ug/mL, all from Becton Dickinson, Mountain View, CA.

Coelomocytes were labeled by incubation of 2x lo6 cells in 50 uL of BSS 0.02% sodium azide (20 min at 4°C) with the appropriate concentration of the corresponding mAb or the isotype control. After two washes with BSS, cells were fixed with 0.2% paraformaldehyde, transferred to FACScan tubes and analyzed. Labeling was performed both on unstimulated coelomocytes and after 72 h stimulation to detect changes on cells. Stimulation was performed with LPS either in the presence or in the absence of plasma as described for the phagocytosis assay.

To evaluate the relationship between phagocytic activity and CD14-like expression, coelomocytes were allowed to phagocytose FITC-HKSC and were further labeled with PE-Leu-M3 mAb. In this double fluorescence assay cells were not quenched with Trypan Blue dye.

Flow Cytometry

A FACScan (Beckton Dickinson Im- munocytometry Systems, San Jose, CA) was used to detect FITC and PE fluorescence. Fluorescence parameters from single cells were collected using a logarithmic amplifier after gating on the combination of forward light scatter (FSC) and perpendicular light scatter (SSC). Red fluorescence from PE was collected through the FL2 channel and green fluorescence from FITC through the FL1 channel. A total of 10 000 cells was analyzed per tube and data acquired in list mode were processed using either Consort 32 Lysis II software or Multi2D (Phoenix Flow, San Diego, CA). The


fluorescence distribution was displayed as a single histogram or two color dot plot analysis. The percentage of fluorescent cells was determined in each case.

Statistical Analysis

The percentage of phagocytic cells or positively stained cells under different stimulating conditions were reported as meanfSEM, and were analyzed using the two-tailed unpaired Student’s t-test. Data with p < 0.01 were considered significant.

Results

FACScan Analysis of Coelomocytes

Several preliminary assays were performed to characterize the FSC and SSC distribution of coelomocytes. Slight variations were observed depending on the month of the year when the animals were collected. Also, the ratio of female to male and the size of individual animals used in the pooled sample of coelomic fluid led to minor differences in the FSC and SSC distribution observed. Figure la shows a FSC versus SSC dot plot of coelomocytes after 72 h culture in RPM1 representative of the distribution most often observed, and Figure 1 b the auto- fluorescence of gated events. The FSC- SSC distribution of coelomocytes incubated with FITC-HKSC in a 1:l target/ effector ratio is shown in Figure lc.

Phagocytic coelomocytes were identified by FITC fluorescence after quenching with Trypan Blue dye and the percentage of these cells in each sample was evaluated as shown in Figure Id. The reverse gating technique was used to identify the FSC and SSC distribution of phagocytic coelomocytes. The dot plot diagram shows that phagocytic coelomocytes are predominantly small and slightly granular, but some larger and more granular cells are also present (Fig. If).

For the phagocytosis assays, gating on FSC and SSC was defined to include all phagocytic coelomocytes as shown in Figure 1. Reverse gating of the unquenched sample shows that the gate excludes a large proportion of uningested FITC-HKSC (Fig. le) since these cells have low FSC and SSC.

Phagocytosis Assay

Ejkct of pre-incubation of coelomocytes with LPS, polymixin B and PMA for 20 h. Coelomocytes pre-incubated either with 1.5 ug/mL PMA or 5 ug/mL LPS for 20 h were stimulated to phagocytose FITC-HKSC as compared to phagocytic values in RPM1 alone (p < 0.00 1). Coelo- mocytes exposed to 10 pg/mL polymixin B showed a lower phagocytic level which was similar to that observed with medium alone. In addition, the presence of polymixin B blocked the LPS-induced stimulation of phagocytosis (Fig. 2a).

Ejkct qf pre-incubation qf coelomocytes with LPS in the presence or absence qf coelomic plasma during 72h. Coelomo- cytes pre-incubated with 2 ug/mL of LPS in the presence of plasma showed higher percentage of coelomocytes phagocytosing FITC-HKSC when compared to values for coelomocytes pre-incubated in RPM1 alone or RPM1 plus coelomic plasma without LPS (p < 0.001). Coelo- mocytes pre-incubated in the presence of 2 ug/mL of LPS without plasma showed phagocytic values higher than controls (p<O.OOl) but lower than those observed when plasma was simultaneously present (p < 0.01) as shown in Figure 2b.

Labeling of Coelomocytes

Unstimulated, freshly extracted coelomocytes showed variable levels of labeling with the anti-human CD14 mAb Leu-M3. Positive cells were approximately 40% in

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Flgure 1. (a) FSC-SSC dot plot of coelomocytes after 72 h of culture in RPMI. Gating defined on FSC and SSC parameters to determine the percentage of coelomocytes phagocytosing FITC-HKSC. (b) auto- fluorescence of gated events. (c) FSC-SSC dot plot of coelomocytes exposed to FITC-HKSC and gated as shown in (a). (d) FITC fluorescence (FLl-H) of gated events (sample quenched withTrypan Blue dye). Number in quadrant 4 indicates percentage of phagocytic coelomocytes. (e) FSC-SSC dot plot of fluorescent cells (reverse gatlng) of unquenched sample, corresponding either to phagocytic coelomocytes or to uningested FITC-HKSC. The gate shown is the same as (a) and (c). (f) FSC-SSC dot plot of fluorescent cells (reverse gating) after quenching withTrypan Blue.The gate shown is the same as above.


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Flgure 2. (a) Phagocytosis of FITC-HKSC by coelomocytes pre-incubated for 20 h either with medium alone (RPMI), with 1.5 pg/mL PMA, with 5 FglmL LPS, with 50 pg/mL polymixin B (PMB) or with 50 pg PMB together with 50 pg/mL LPS (PMB + LPS). Vertical bars indicate mean f 2- SEM. Asterisks indicate significant difference from value for coelomocytes incubated in RPM1 alone (p<O.OOl determined by unpaired t-test; N = 3). (b) Phagocytosls of FITC-HKSC by coelomocytes pre-incubated for 72 h with either medium alone (RPMI), medium containing coelomic plasma diluted l/20 (PLASMA), 2 PglmL LPS or 2 pg/mL LPS plus coelomic plasma diluted l/ 20 (LPS + PLASMA). Vertical bars indicate mean f’2SEM. Single asterisks indicate significant difference from value for coelomocytes incubated in RPM1 alone (p < 0.001 determined by unpaired f-test; N = 4). Double asterisk indicates significant difference from value for coelomocytes incubated with 2 pg/mL LPS without plasma (p < 0.01; N = 4).

ungated samples (Fig. 3a). Background fluorescence was detected with coelomocytes labeled with isotype control antibody (Fig. 3b). When reverse gating was applied, the FSC and SSC dot plot distribution of anti-CD14 positive coelomocytes showed predominantly small and slightly granular cells, although some larger and more granular cells were also present (Fig. 3~).

Neither anti-human CD1 la nor anti- human CD 18 were able to identify surface markers on sipunculan coelomocytes (Fig.

3d-f). However, anti-CD1 lb mAb Leu- 15 and anti-CD1 lc mAb FHCL-3 identified a high proportion of coelomocytes as shown in Figures 3e and 3f, respectively.

When coelomocytes were incubated for 72 h with either medium alone, medium containing coelomic plasma diluted l/20, 2 pg/mL LPS, or 2 pg/mL plus coelomic plasma diluted l/20, and further labeled with anti-CD1 lb or anti-CDllc mAbs, differences in the percentage of positive coelomocytes were observed. Figure 4a and 4b show results of labeling coelomocytes with anti-CD1 lb and anti-CD1 1 c, respectively. In both cases the highest values were obtained when both plasma and LPS were present in the culture media (p < 0.001 compared to values for coelomocytes in RPM1 alone). However, in the absence of LPS the percentage of either anti-CD1 1 b positive or anti-CD 1 lc positive coelomocytes was also higher when incubated in the presence of plasma rather than medium alone (p < 0.001).

No significant differences were observed in the percentage of anti-CD14 positive coelomocytes when incubated in 2 pg/mL LPS with or without plasma for 72 h (data not shown).

Phagocytosis and CDI4-like Labeling

To evaluate the relationship between CD 1Clike labeling and phagocytosis, coelomocytes were allowed to ingest FITC-HKSC and were further labeled with mAb Leu-M3.

Double fluorescence was associated with anti-CD 14 positive coelomocytes phagocytosing FITC-HKSC (15.15%), whereas PE positive-FITC negative cells were non-phagocytic anti-CD14 + coelomocytes (10.18%). FITC positive-PE negative cells (29.40%) were either anti- CD 14 negative phagocytic coelomocytes or cells associated with uningested FITC- HKSC, since these were not quenched (Fig. 5a). FITC-HKSC were not labeled by mAb Leu-M3 (Fig. 5b). FSC-SSC


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Figure 3. (a) FL1 versus FL2 dot plot of an ungated sample of coelomocytes labeled with PE coupled anti-human CD14 mAb Leu-M3. (b) FL1 versus FL2 dot plot of an ungated sample of coelomocytes labeled with isotype control (lgG2b). (c) FSC versus SSC distribution of CD14 positive cells (reverse gating). (d) FL1 versus FL2 dot plot of coelomocytes labeled with PE coupled anti-human CDlla. (e) FL1 versus FL2 dot plot of coelomocytes labeled with PE coupled anti-human CDllb and FITC coupled anti-human CD18 (ungated sample). (f) FL1 versus FL2 dot plot of coelomocytes labeled with PEcoupled anti-human CDllc and FITC coupled anti-human CD18 (ungated sample). Numbers indicate percentage of positive cells.


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distribution of phagocytic anti-CD 14 positive coelomocytes showed that these cells were mainly small and slightly granular, although large and more granular cells were also present (Fig. 5d).

Effect of PI-PLC treatment

Coelomocytes treated with PI-PLC showed a decrease in the percentage of anti-CD14 positive cells to about 30% of that of untreated samples, as judged by the evaluation of samples gated as shown in Figure 6. This result indicates that the sipunculan CDlClike antigen may be anchored by phosphatidyl-inositol linkage in a great proportion of coelomocytes.

Discussion

Results of our experiments indicate that phagocytosis of FITC-HKSC by coelomocytes of Them&e petricola is enhanced by LPS and PMA. These results are in agreement with observations of the effects of LPS in the immune systems of other invertebrate animals. LPS has been proposed to serve as a hemocyte activating signal in molluscs in case of infection by Gram- negative bacteria (33). It has been demonstrated that LPS induces the stimulation of migratory activity in hemocytes from Mytilus edulis (33), and the stimulation of cellular conformational changes in immunocytes of mol- lusts Planorbarius corneus, Mytilus edulis and the insect Leucophaea maderae (34,35). LPS and PMA have also been shown to stimulate hemocyte aggrega- tion in the silk moth Hyalophora cecropia, and it has been demonstrated that the bacteria-inducible protein hemolin synergizes with LPS in the enhancement of phagocytosis. It has been suggested that hemolin is involved in the regulation of the cellular immune response of insects via a pathway that includes protein kinase C activation and protein tyrosine phosphorylation (36). Evidence from work in crustaceans, some species of insects and other invertebrates has shown that the so- called pro-phenoloxidase system is turned into its active form by the presence of either LPS, B-1,3-glucans or peptidoglycans derived from fungal cell walls. Proteins associated with the prophenoloxidase system have been shown to promote phagocytosis and capsule formation around parasites (37).

Blockade of LPS-induced stimulation of phagocytosis in Themiste petricola by polymixin B indicates that coelomocyte binding of LPS is a necessary and sufficient condition for the observed effect. Polymixin B has also been shown to prevent LPS-induced activation of


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Figure 5. (a) Coelomocytes were allowed to phagocytose FITC-HKSC and further labeled with mAb Leu-MS. Quadrant 1 shows non-phagocytic CD14 positive coelomocytes (upper left); quadrant 2 shows phagocytic CD14 positive coelomocytes (upper right); quadrant 4 shows either phagocytic CD14 negative coelomocytes or uningested FITC-HKSC (lower right) and quadrant 3 shows non-phagocytic CD14 negative coelomocytes (lower left). The horizontal line was placed by use of negative control (PE-labeled isotype control) as shown in (c). (b) Free unquenched FITC-HKSC were not identified by mAb Leu-M3. (c) Coelomocytes were allowed to phagocytose FITC-HKSC and labeled with PE-labeled isotype control (IgG2b). The isotype control did not identify coelomocytes. FL1 fluorescence corresponds either to uningested FITC-HKSC or to coelomocytes phagocytosing FfTC HKSC. (d) FSC-SSC dot plot of phagocytic CD14 positive coelomocytes [upper right quadrant in (a)]. Numbers in figures (a)-(c) indicate percentage of cells.

human and murine leukocytes when co- incubated with LPS in the absence of serum (38). A stoichiometric relation between LPS and polymixin B in a serum free buffer system has been reported by Morrison and Jacobs (39), suggesting the binding of polymixin B to one monomer unit of LPS.

Our results show that, when LPS was present in the culture medium together

with coelomic plasma diluted l/20, the maximal rates of phagocytosis are observed. Based on the role of LBP in mammalian immune system, where this molecule acts as a LPS opsonin or carrier, increasing the stimulating potency of the same dose of LPS (21,24-27), we could hypothesize about the presence of a LBP- like molecule in coelomic plasma of Themiste petricola, with similar functions.


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Figure 6. Effect of PI-PLC treatment on the expression of the sipunculan CD14 cross-reactive molecule. (a) Gating of coelomocytes; (b) CM4 positive coelomocytes of untreated samples; (c) CD14 positive coelomocytes In PI-PLC treated sample. Numbers indicate percentage of cells.

However, the presence of such a mechanism to explain the observed results requires further elucidation.

Our search for sipunculan cross-reactive molecules with anti-human PZinteg- rin mAbs has revealed the presence of CD 11 b- and CD 1 lc-like molecules, although we failed to detect a CDl%like molecule. Variations observed in the percentage of anti-CDllb and anti- CD1 lc positive in vitro stimulated coelomocytes correlated with the observations in the phagocytosis assays, suggesting that they could have a role in defense mechanisms of sipunculans. However, it remains to be elucidated whether or not the sipunculan cross-reactive antigens are

related to the c1 chain of CR3 and pl50,95 at the molecular level, and if functions ascribed to these mammalian molecules are also present. Interestingly, CR3 has important functions as an adhesion molecule in mammals and was recently characterized as a phagocytosis receptor with specificity for P-glucan residues (40- 43). LPS can bind to and activate mammalian CR3, enhancing its ability to mediate phagocytosis (44).

Since sipunculan coelomocytes showed no cross-reactivity to the CD1 la and CD1 8 antigens, it is interesting to contrast these results to recent data obtained in earthworm leukocytes. Using a broad panel of mouse anti-human mAbs, Cos-


sarizza et al. reported cross-reactivity with CD 11 a, CD45RA, CD45R0, CDw49b, CD54 and l&m in earthworm leukocytes. Only non-phagocytic small electron-dense cells with NK-like activity were positive for these markers, while phagocytic large electron-lucent cells were negative. In the same work, cross-reactivity was not detected with CD14, CD1 lb, CD18 and HLA-DR (45).

Finally, a human CD14 cross-reactive molecule was detected with mAb Leu-M3 on the surface of sipunculan coelomocytes. We have obtained similar results with another anti-human CD14 mAb (UCHMl, data not shown). Our finding that phagocytic coelomocytes are mainly anti-CD14 positive, as shown in the double fluorescence assay, indicates that this antigen is present in a coelomocyte subtype directly involved in sipunculan defense functions. The fact that a significant reduction in percentage of anti- CD14 positive coelomocytes was observed after incubation with the enzyme PI-PLC suggests that this cross-reactive molecule may be anchored to the cell membrane by a phosphatidyl-inositol linkage similar to the mammalian CD14. Although enzyme treatment did not remove all positive coelomocytes, it has

been shown by other authors that treatment of mammalian cells with PI-PLC also fails to remove CD14 from some cells, possibly owing to conformational changes or chemical substitutions that reduce the accessibility of the enzyme to the hydrolysis site (46). It would be interesting to determine if any of the functions currently known for the mammalian CD14 are also associated with the sipunculan cross-reactive molecule.

We conclude that LPS upregulated the phagocytic response and the expression of antigens cross-reactive with human CD1 1 b and CD1 lc in sipunculan coelomocytes. Our studies have been performed based on the current knowledge of the effects of LPS on mammalian systems. However, results have shown similarities to the situation observed in the phylogenetically close phylum Mol- lusca and other invertebrate groups. It would be interesting to determine whether or not homology to the mammalian molecules is present in any of the cross-reactive molecules. We are now evaluating the functional role of sipunculan CD14-, CDllb- and CDllc- like molecules to investigate their participation in the defense system of Them&e petricola.

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LPS-induced stimulation of phagocytosis in the sipunculan worm Themiste petricola: Possible involvement of human CD14, CD11B and CD11C cross-reactive molecules