Resistance Against the Membrane Attack Complex of Complement Induced in Porcine Endothelial Cells with a Galα(1–3)Gal Binding Lectin: Up-Regulation of CD59 Expression

Reagents

DMEM, RPMI 1640, HBSS with Ca/Mg, l-glutamine, FBS, and antibiotics were purchased from Life Technologies (Grand Island, NY). BS-I and Lycopersicon esculentum lectin were obtained from Sigma (St. Louis, MO) or Vector Laboratories (Burlingame, CA). IB₄ and other lectins were obtained from Vector Laboratories. Phalloidin-tetramethylrhodamine isothiocyanate (TRITC), oligosaccharides, cycloheximide, melittin, streptolysin O, neutral red, detergents, and general chemicals were obtained from Sigma. Goat anti-BS-I was obtained from Vector Laboratories, and HRP-conjugated, affinity-isolated, polyclonal Abs against human IgG, human IgM, or goat IgG were obtained from Tago (Burlingame, CA). Mouse IgG mAbs against C3bi or C9 neoantigen and C8-depleted human serum were obtained from Quidel (San Diego, CA), and goat antisera against human C4 and C6 were obtained from Organon Teknika (Capel, Durham, NC). A mouse IgG mAb against porcine CD59 (MEL-3) was a generous gift of Dr. B. P. Morgan (University of Wales, Cardiff, U.K.). Affinity-isolated, HRP-conjugated, goat anti-mouse IgG and Fluoromount-G were obtained from Southern Biotechnology Associates (Birmingham, AL). Mouse IgG mAb against human E-selectin (BBIG-E6) was obtained from R&D Systems (Minneapolis, MN), and o-phenylenediamine was obtained from Abbott Laboratories (North Chicago, IL). Human C8 was purified in this laboratory (32). Kits for PGI₂ measurement as the stable metabolite 6-keto-PGF_1α were obtained from Cayman Chemical (Ann Arbor, MI). Radioisotopes were obtained from NEN Life Science Products (Boston, MA).

Endothelial cells

EC were explanted from porcine aortae, cultured, and identified as previously described (33, 34, 35). Cells were maintained in DMEM with l-glutamine, antibiotics, and 10% FBS (29). Experiments were performed with cells in passages 4–12, 3 days postconfluent in gelatin-coated tissue culture plates, as follows: plates with 96 wells for ELISA, 48 wells for cytotoxicity and metabolic assays, and 24 wells for PGI₂ quantitation (Costar, Cambridge, MA), 8-well glass chamber slides for morphology studies, and 100-mm tissue culture dishes for mRNA isolation (Becton Dickinson, Franklin Lakes, NJ). All incubations were conducted at 37°C in a 5% CO₂ atmosphere.

Source of anti-pig natural Abs and C

Two pools of normal human serum were prepared such that they had normal C levels and either a high titer (serum A) or a low titer (serum B) of anti-porcine EC Abs as determined by ELISA (36). Serum A was incubated at 56°C for 30 min to inactivate C, stored at −70°C, and used as a source of natural Abs. Serum B had a level of IgM natural Abs that was low but sufficient to trigger EC cytotoxicity and was used as the source of natural Abs and C for all cytotoxicity assays (29). Ig-depleted human serum was prepared by affinity chromatography, and IgM was purified from serum A and evaluated for purity, stored, and dialyzed as described (29). Rabbit serum from an individual animal and a pool of rat sera were used as the respective complement sources.

Treatment of EC with lectin and measurement of cytotoxicity

EC were preincubated with 250 μl of heat-inactivated serum or a lectin in DMEM containing 5% FBS. In some experiments, the lectin solution was removed, the cells were washed twice with DMEM-5% FBS, then 250 μl of the same medium was added and the preincubation continued. When preincubation was completed, the cells were washed twice with RPMI 1640 containing 2% human serum albumin. The number of EC after lectin-treatment was assessed by neutral red uptake (37) before exposure to Abs and C or performance of an ELISA. Neutral red uptake by lectin-treated EC was within ± 6.5% of uptake by medium-treated control EC, indicating that their viability had not been impaired (37). This assessment was validated by microscopic counts of EC in 96-well plates after fixation as for ELISA and staining with Giemsa-Wright. Cell counts (×10³ cells/well, mean ± SE of triplicate wells) in experiment 1 were 6.7 ± 0.14 for EC treated with 100 μg/ml BS-I and 6.7 ± 0.16 for controls, and in experiment 2 cell counts were 8.8 ± 0.14 for BS-I-treated EC and 8.9 ± 0.09 for controls. Similar numbers were obtained when 25 μg/ml BS-I was used.

EC cytotoxicity was measured with a vital dye assay (37). Pretreated cells were incubated for 4 h with 150 μl serum in RPMI 1640 or for 2 h with 150 μl PBS containing 1.5 μM melittin or 2000 U streptolysin O activated with 10 mM DTT for 5 min at room temperature (RT). The cells were then washed twice with HBSS. After addition of 250 μl neutral red at 67 μg/ml in DMEM-10% FBS, the cells were incubated for 2 h, washed with HBSS, and mixed with 250 μl of 1:1 (v/v) 1% acetic acid-ethanol. The plate was shaken for 10 min to dissolve the dye, 100 μl were transferred to a 96-well plate, and the OD540 was determined with a Vmax microplate reader (Molecular Devices, Sunnyvale, CA). Percent specific cytotoxicity was calculated after correction for the OD of solubilized cells not treated with the dye as follows: % cytotoxicity = [1 − (test OD/control OD)] × 100. Values are given as mean ± SE of triplicate samples or mean and range of duplicate samples.

To determine whether C treatment caused a functional membrane channel, the radioactivity released from ⁸⁶Rb- and ⁵¹Cr-labeled EC was measured. Preincubation with BS-I and washes were performed as described above. Release was determined from quadruplicate wells with pretreated EC that were labeled at 37°C with either ⁵¹Cr in HBSS for 2 h (36) or ⁸⁶Rb in RPMI-5% FBS for 1 h (38). The cells were washed, incubated with 40% serum B in RPMI 1640 from 2 min to 3 h, and radioactivity was counted in supernatants and cell monolayers. Percent specific release was calculated as described, after correction for spontaneous marker release (36). To investigate αgal binding specificity for BS-I-induced C resistance, EC were treated for 4 h with BS-I in DMEM-5% FBS containing either 3-α-galactobiose, melibiose, or lactose. The cells were washed with DMEM-5% FBS, 250 μl of the same medium was added, and the preincubation was continued for 16 h. To determine whether lectin treatment affects the metabolic activity of EC, the cells were treated with 100 μg/ml BS-I in DMEM-5% FBS for either 4 h or 20 h, then washed and further incubated for 0–48 h in medium alone. The medium was removed and 250 μl of [³H]thymidine at 5 μCi/ml in DMEM-10% FBS was added (39) followed by 18 h incubation at 37°C. The cells were washed five times with HBSS-1% FBS at 4°C and treated with 250 μl cold TCA (10% w/v) for 5 min. The precipitate was washed with cold 8% TCA, solubilized with 250 μl 0.2 N NaOH/0.2% SDS, and radioactivity was counted to assess incorporation of [³H]thymidine into DNA.

Release of PGI₂

EC were preincubated in 24-well plates with 500 μl BS-I in DMEM-5% FBS for 20 h. The cells were washed with DMEM-5% FBS and incubated with 250 μl 25% heat-inactivated serum as a source of Abs, washed with HBSS, and then incubated for 30 min at 37°C with 250 μl of the C source diluted in HBSS. The supernatants were collected, centrifuged at 2000 × g, and PGI₂ was determined by ELISA of its stable metabolite 6-keto-PGF_1α (40).

Binding of Ab, C components, and BS-I

EC were preincubated with 100 μl BS-I in DMEM-5% FBS for 1 h or 24 h. After preincubation, the cells were washed twice with RPMI 1640, incubated for 1 h with 50 μl of 40% serum B in RPMI 1640, and then binding of Ab and C components was measured in triplicate wells by ELISA (29). The cells were washed twice with PBS-0.1% BSA, twice with PBS alone, fixed with 0.1% glutaraldehyde, and, after 3 washes in PBS, blocked with 250 μl 1.0% BSA in PBS for 1 h at RT. Binding of IgG or IgM was detected with the corresponding HRP-conjugated anti-human Ig Abs. Deposition of C3bi, C4b, C6, and C9 on the EC was detected with the respective specific Ab followed by HRP-conjugated, anti-IgG (4). Then, 50 μl Ab in PBS-1.0% BSA was added to the cells for 1 h at RT. Reaction product was generated with o-phenylenediamine and assayed at 405 nm. Binding of BS-I was assessed on EC that were incubated with 100 μl BS-I in DMEM-5% FBS from 1 min to 30 min or for 4 h. EC that were treated for 1–30 min were immediately prepared for ELISA as above. EC that were treated for 4 h were washed with DMEM-5% FBS, then 100 μl DMEM-5% FBS was added and the incubation continued for 4–60 h; the cells were then prepared for ELISA. Bound lectin was assessed with goat anti-BS-I followed by HRP-conjugated anti-goat IgG. Then, 50 μl Ab in PBS-1.0% BSA was added for 1 h at RT, and the reaction product was measured.

Expression of E-selectin

EC were preincubated with 100 μl BS-I in DMEM-5% FBS for 4 h; after 2 washes with DMEM-5% FBS, 100 μl DMEM-5% FBS was added, and the preincubation continued for 1 h to 16 h. The cells were washed twice with PBS-0.1% BSA, twice with PBS alone, then fixed with 0.03% glutaraldehyde and blocked with 1.0% BSA as above. E-selectin was detected with mouse IgG mAb against human E-selectin that cross-reacts with porcine E-selectin (26) followed by HRP-conjugated, goat anti-mouse IgG. Then, 50 μl Ab in PBS-1.0% BSA was added to the cells for 1 h at RT. The reaction product was generated and assayed as above.

Expression of CD59 mRNA

Total RNA was isolated from EC cultured in 100-mm dishes at various times following incubation at 37°C with 10 ml of 50 μg/ml BS-I in DMEM-5% FBS or medium alone. Washed EC were lysed with 3 ml of TRIzol reagent, and RNA was isolated as described by the manufacturer (Life Technologies). For Northern blot analysis, 10 μg of total RNA were separated on 1.2% (w/v) agarose gels containing 6.7% (v/v) formaldehyde, 1 mM EDTA, 20 mM sodium phosphate, pH 7.0, and 5 mM sodium acetate, and transferred to nitrocellulose using 20× SSC. The blots were hybridized as previously described (41) using a gel-purified pig CD59 cDNA insert (42). The amounts of RNA in loaded samples were normalized by hybridization with a β-actin cDNA insert (43). The amount of CD59 and β-actin signal was determined using a phosphorimaging system and ImageQuant software (Molecular Dynamics, Foster City, CA).

Expression of membrane-associated CD59

EC were preincubated with 100 μl BS-I in DMEM-5% FBS for 4 h or 20 h and then prepared for ELISA as described for assessment of E-selectin expression. CD59 was detected with a mouse IgG1 mAb against porcine CD59. Then, 50 μl mAb at 0.4 μg/ml in PBS-1.0% BSA was incubated with the cells for 1 h at RT. Nonspecific binding of mouse IgG was assessed with purified mouse IgG1 myeloma protein (Harlan Bioproducts, Indianapolis, IN), and these values were subtracted from those with the anti-CD59 mAb. Bound Ab was detected with HRP-conjugated, goat anti-mouse IgG as above.

Visualization of actin filaments

EC were preincubated with BS-I in 200 μl DMEM-5% FBS at 37°C for 4 h or 20 h. From some wells, the BS-I solution was removed, the cells were washed twice with DMEM-5% FBS, 200 μl of this medium was added, and the incubation was continued for 24 h or 48 h. The supernatants were removed from all wells, and the EC were washed three times with cold (4°C) PBS-1.0% FBS and once with cold PBS alone. The cells were then fixed with 3.5% paraformaldehyde for 5 min at RT, washed three times with cold PBS-1.0% FBS, once with cold PBS, and permeabilized with 400 μl of 0.1% Triton X-100, 0.5% Nonidet P-40 in PBS for 5 min at RT. Following three washes with 0.1% Tween 20 in PBS, actin was labeled with phalloidin-TRITC (27, 44), 100 μl at 5 μg/ml in PBS-1% FBS-0.1% Tween 20 for 30 min at RT, then washed three times with PBS-0.1% Tween 20. Slides were mounted with Fluoromount G and imaged at 200× using a Zeiss Axioplan fluorescence microscope, with mercury lamp sample excitation and a rhodamine filter. Photomicrographs were taken with a digital Photometric camera (Photometrics, Tucson, AZ) using an Intel-driven computer and Metamorph software (Universal Imaging, West Chester, PA).

B. simplicifolia lectin induces resistance of porcine EC against C-mediated cytotoxicity

We initially determined the ability of BS-I to induce resistance of EC to cytotoxicity by human C in comparison to anti-pig natural Abs (29). After incubation with BS-I or heat-inactivated serum as a source of natural Abs during various time periods, the efficacy of resistance induction was tested by exposure to human serum as a source of natural Ab and C. Fig. 1⇓A shows that BS-I, in comparison with natural Abs, is highly effective in inducing resistance. However, a similar degree of resistance was induced when similar amounts of agonists were used for 20 h or 40 h, either 22 nM BS-I (2.5 μg/ml) or 24 nM anti-pig IgM natural Abs (40% human serum, the level of anti-pig IgM natural Abs was estimated as 3% of total IgM; Ref. 45) (results not shown). We then investigated whether BS-I-treated cells are protected from C-mediated cytotoxicity over a range of the cytotoxic activity in human serum. BS-I-treated cells were tested with Ig-depleted serum containing normal C levels and reconstituted with increasing amounts of purified human IgM as a source of natural Abs. Fig. 1⇓B demonstrates that BS-I-treated cells were protected when exposed to degrees of C activation that caused increasing levels of cytotoxicity in control cells. This protection was not limited to human C as lectin-treated EC also exhibited resistance to killing by rat or rabbit C (Table I⇓). To ascertain whether the protection is specific for C, we evaluated the susceptibility of EC to lysis by the pore-forming agents melittin and streptolysin O. Results show that lectin-treated cells were not protected against either agent, suggesting that the lectin-induced protection is specific for C (Table I⇓).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

Induction of resistance against C-mediated cytotoxicity in EC by stimulation with B. simplicifolia lectin. A, Comparison of the effect of BS-I with anti-pig natural Abs on susceptibility of porcine EC to C-mediated killing. EC were preincubated at 37°C with 50 μg/ml BS-I, 25% heat-inactivated human serum as a source of natural Abs (NAb), or with medium alone during the time periods indicated. The cells were then washed and incubated for 4 h at 37°C with 40% human serum as a source of natural Abs and C. In this and subsequent experiments, cytotoxicity was measured with a neutral red vital dye assay. Results are representative of three independent experiments. B, Lectin-treated EC are protected against C-mediated cytotoxicity over a wide range of cytotoxic activity of a human serum. The cells were preincubated for 20 h at 37°C with 50 μg/ml BS-I or with medium alone, washed, and incubated for 4 h at 37°C with 50% Ig-depleted human serum that was reconstituted with various doses of purified human IgM containing anti-pig natural Abs. Cytotoxicity was then measured. Results are representative of two similar experiments.

Induction of resistance to C by B. simplicifolia lectin requires binding of the lectin to αgal

A dose-response study using BS-I and IB₄ demonstrated that the effect is dose dependent; nearly complete resistance was obtained with 50 μg/ml BS-I or 25 μg/ml IB₄ (Fig. 2⇓A). BS-I is a heterogeneous tetramer of two subunits; one binds only terminal αgal, the other binds αgal and also N-acetylgalactosamine; IB₄ is a tetramer of the αgal-specific subunit. Fig. 2⇓A shows that IB₄ was more efficient than BS-I in causing resistance, suggesting that induction of resistance is αgal mediated. Induction of resistance was blocked by incorporation of melibiose or galactobiose in the lectin incubation step but not with lactose, demonstrating that the induction requires binding of the lectin to αgal-containing receptors (Fig. 2⇓B). We also tested induction of resistance with lectins that bind sugars other than αgal (46). Although these lectins modified somewhat the EC susceptibility to C, they failed to induce resistance (Fig. 2⇓C). Of special interest was that Vicia villosa lectin failed to elicit resistance. As V. villosa lectin binds to N-acetylgalactosamine, one of two oligosaccharides to which BS-I A chain binds, this result also suggests that BS-I induces resistance through its capacity to bind αgal, not N-acetylgalactosamine.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

Induction of resistance of porcine EC against C-mediated killing requires binding of B. simplicifolia lectin to αgal epitopes. A, Dose-response relationship in induction of resistance to C-mediated killing of EC preincubated with lectins BS-I or IB₄. EC were incubated with the lectins at 37°C for 20 h, washed and incubated for 4 h at 37°C with 40% human serum as a source of natural Abs and C, and cytotoxicity was measured. Results are representative of three similar experiments. B, EC were preincubated for 4 h at 37°C with 50 μg/ml BS-I or with medium alone in the presence of galactobiose (αgal(1–3)gal), melibiose (αgal(1–3)glu), or lactose (βgal(1–4)glu); the cells were then washed and incubated with medium alone for 16 h. Finally, the cells were washed and incubated for 4 h at 37°C with 40% human serum as a source of natural Abs and C, and cytotoxicity was measured. Results are representative of two similar experiments. C, EC were preincubated for 20 h at 37°C with 50 μg/ml lectin or medium alone. The cells were then washed and incubated for 4 h at 37°C with 40% human serum as a source of Abs and C, and cytotoxicity was measured. Results are representative of three similar experiments. The specificity of the lectins is as follows: Lycopersicon esculentum lectin (LEL), N-acetylglucosamine; Maackia amurensis lectin-I (MAL-I), βgal(1–3)N-acetylglucosamine; Vicia villosa lectin (VVL), N-acetylgalactosamine; Lens culinaris lectin (LCL), mannose; and BS-I, αgal and N-acetylgalactosamine.

αGal-binding lectin elicits protection in EC from the stimulation of PGI₂ secretion by sublytic amounts of MAC

Because it is known that the MAC in sublytic amounts activates several proinflammatory cell processes, we investigated whether MAC-induced PGI₂ secretion is impaired in EC following treatment with BS-I. Results in Fig. 3⇓ show that pretreatment of EC with BS-I abrogated the release of PGI₂ in response to stimulation with two human sera used as a source of natural Abs and C. A control consisting of C8-depleted serum failed to induce PGI₂ secretion from EC not exposed to BS-I; restoration of PGI₂ secretion was achieved by reconstitution of this serum with purified C8, confirming that the effect of C is due to the MAC.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

Incubation of porcine EC with B. simplicifolia lectin impairs their ability to release PGI₂ upon stimulation with a sublytic dose of C. EC were incubated at 37°C for 20 h with 100 μg/ml BS-I or with medium alone, washed, and then incubated at 37°C for 30 min with 25% heat-inactivated human serum as a source of natural Abs. The cells were then washed and incubated for 30 min at 37°C with two human sera at 15% as C sources. Results with C8-depleted human serum at 25% (C8D serum) and C8D serum reconstituted with 50 μg/ml C8 confirmed that release of PGI₂ is MAC dependent. Results are representative of three experiments.

Pretreatment of EC with lectin does not interfere with binding of IgM natural Abs and C components but the MAC channel is defective

Because both BS-I and anti-pig natural Abs bind αgal, we asked whether pretreatment with BS-I inhibited binding of natural Abs. We measured cell-bound proteins after 1 h of incubation with the C source, before cytotoxicity occurred. EC that had been preincubated with BS-I bound somewhat less IgG natural Abs than cells treated only with medium; however, pretreatment with BS-I for 1 h or 24 h did not interfere with binding of IgM natural Abs (Fig. 4⇓). Binding of C3bi, C4, C6, and C9 by EC that were pretreated with BS-I was similar to or better than in control cells (Fig. 4⇓); EC-bound BS-I did not trigger C activation in the absence of Ab (results not shown). Therefore, suppression of C-mediated cytotoxicity in lectin-treated EC is not due to inhibition of Ab binding, C activation, or deposition of MAC proteins on the cell surface.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

Binding of IgG and IgM anti-pig natural Abs and C components by porcine EC pretreated with B. simplicifolia lectin. EC were preincubated at 37°C with 50 μg/ml BS-I (solid line) or with medium alone (dashed line) during 1 h or 24 h. The cells were washed and incubated for 1 h at 37°C with 40% human serum containing C and a low level of IgM anti-pig EC natural Abs. Binding of natural Abs and C components was determined by ELISA. Results are representative of three experiments.

To ascertain whether C5b-8 and C5b-9 induced functional channels when deposited on lectin-treated EC we measured marker release from EC that were labeled with ⁸⁶Rb or ⁵¹Cr. There was a slight reduction in ⁸⁶Rb release from cells that had been preincubated with BS-I (Fig. 5⇓A). However BS-I treatment induced a marked inhibition of ⁵¹Cr release, similar to the inhibition of killing assessed by the uptake of neutral red vital dye (Fig. 5⇓B). Because the EC monolayers had a large background leakage of ⁸⁶Rb (see Fig. 5⇓), we were unable to measure release for >20 min after initiation of C attack. These experiments suggested that BS-I-treated EC have a functional C channel of sufficient size to allow passage of a small marker such as ⁸⁶Rb (diameter = 0.46 nm) but not ⁵¹Cr-labeled EC proteins.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

Modifications in C-induced membrane permeability of porcine EC by pretreatment with B. simplicifolia lectin. The EC were incubated at 37°C with 100 μg/ml BS-I or medium alone for 20 h. The cells were then washed, labeled with ⁸⁶Rb or ⁵¹Cr, and incubated for various times at 37°C with human serum as a source of Abs and C. Marker release was then measured and expressed as indicated in Materials and Methods after correction for spontaneous release. For comparison, uptake of neutral red was also determined. Background marker loss was similar with BS-I-treated EC and controls, 31–39% at 10 min and 44–58% at 20 min for ⁸⁶Rb, and <1.2% at 1 h and <6.5% at 3 h for ⁵¹Cr; nonspecific cell death detected with the neutral red assay was 4.2%. Results are representative of five experiments.

Pretreatment of EC with lectin causes up-regulation of CD59 mRNA expression and membrane-associated CD59

Porcine CD59 is known to effectively inhibit human C (42, 47, 48); therefore, we investigated whether incubation with lectin caused up-regulation of CD59 in EC. Northern blot analysis of total RNA isolated from EC incubated with BS-I or medium from 0 h to 20 h is shown in Fig. 6⇓. Exposure to BS-I resulted in increased CD59 RNA expression beginning at 5 h, which increased steadily to almost a 10-fold increase relative to control cells at the conclusion of the experiment at 20 h. The expression of CD59 on the surface of the EC was also markedly enhanced by incubation with BS-I (Fig. 7⇓). The increase in expression of membrane-bound protein preceded that of mRNA, as by 4 h of lectin treatment membrane CD59 was >2-fold that of controls, while at 5 h BS-I caused only a slight increment in CD59 RNA. These results suggest that BS-I induces up-regulation of CD59 expression in porcine EC by multiple mechanisms.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Northern blot analysis of CD59 expression in porcine EC. A, Total RNA was isolated from EC incubated with or without 50 μg/ml BS-I as described in Materials and Methods; 10 μg RNA per lane were separated on 1.2% agarose-formaldehyde gels, transferred to nitrocellulose, and hybridized with a [³²P]-labeled pig CD59 cDNA or β-actin cDNA as indicated. B, The intensity of CD59 and β-actin signal was determined, and the CD59 data were normalized to the β-actin signal in each sample. The data shown are representative of three independent experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Expression of CD59 by porcine EC stimulated with BS-I. EC were incubated for 4 h or 20 h at 37°C with DMEM-5% FBS or with DMEM-FBS plus 50 μg/ml BS-I. After washing, the cells were fixed and CD59 expression was evaluated by ELISA with a mAb against pig CD59. Results are representative of two experiments.

Induction of EC resistance to C does not require continuous incubation with lectin and is long-lasting

We asked how long is it necessary to maintain the incubation with BS-I to elicit protection against C. EC that had been incubated with BS-I for 20 h but not those incubated from 30 min to 4 h demonstrated resistance to cytotoxicity when tested immediately with C (Fig. 8⇓A). In contrast, if EC were pretreated with BS-I from 30 min to 4 h and kept in medium without BS-I to complete 20 h from the beginning of the lectin treatment and then tested with C, resistance was clearly demonstrated (Fig. 8⇓B). The degree of resistance was proportional to the duration of the initial exposure to the lectin, but induction did not require continuous incubation with lectin. Because additional time was needed for the expression of resistance, we assessed the binding of BS-I to EC and then, after various times of incubation in medium alone, measured the BS-I that remained bound. Results indicated that BS-I bound rapidly to the cells, requiring about 30 min to achieve saturation (Fig. 9⇓A). After incubation with BS-I for 4 h and removal of unbound lectin, ∼80% of the bound lectin remained on the cell surface for >60 h (Fig. 9⇓B). We then investigated how long the cells that were pretreated with BS-I for 4 h would retain the refractoriness to C cytotoxicity. The pretreated cells maintained a profound resistance to C for at least 48 h and were still resistant 72 h after BS-I treatment (Fig. 10⇓).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 8.

Continuous incubation with B. simplicifolia lectin is not required for induction of resistance of porcine EC against C. EC were incubated for the indicated time periods at 37°C with 100 μg/ml BS-I. Susceptibility to C-mediated cytotoxicity was tested by incubation for 4 h at 37°C with 40% human serum as a source of Abs and C. In A, the cells were washed and tested immediately for C susceptibility. In B, the cells were washed and incubated with medium alone at 37°C to complete 20 h of incubation before testing with C. Results are representative of three experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 9.

Binding of the lectin BS-I to porcine EC. The cells were incubated at 37°C with BS-I at 25 μg/ml (○) or 100 μg/ml (•) and then washed. A, Bound BS-I was assessed by ELISA immediately after incubation with BS-I for the indicated time periods. B, The cells were incubated with BS-I for 4 h, washed, and then incubated with medium alone for the indicated time periods, after which bound BS-I was determined. Results are representative of three experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 10.

Onset and duration of resistance to C-mediated killing after preincubation of EC with BS-I. EC were incubated for 4 h at 37°C with 100 μg/ml BS-I or with medium. The cells were washed and then incubated with medium alone for the indicated time periods. Susceptibility to C was tested with 40% human serum for 4 h at 37°C. Results are representative of three similar experiments.

Resistant cells display normal morphology and viability

It is known that BS-I induces immediate morphologic changes and actin rearrangement as an early manifestation of EC activation (27). Therefore, we investigated whether these changes occur after incubation with BS-I under conditions that result in resistance to C. Incubation with BS-I for 4 h or 20 h caused profound changes in morphology, with retraction and redistribution to the cell periphery of actin filaments, as shown with fluorescent-labeled phalloidin (Fig. 11⇓). In contrast, if these cells were allowed to recover for 20–48 h following the lectin treatment, they showed a return to normal filament distribution and a substantial recovery toward the morphologic appearance of resting cells (Fig. 11⇓). At this time, the cells were almost completely resistant to C (see Fig. 10⇑), exhibited normal uptake of the vital dye neutral red, and incorporated [³H]thymidine as did the controls, although lectin treatment may initially enhance [³H]thymidine uptake (Table II⇓).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 11.

Reorganization of actin filaments after preincubation of EC with BS-I is reversible. The cells were incubated for various times at 37°C with medium or 100 μg/ml BS-I, then washed and fixed, permeabilized, and stained with phalloidin-tetramethylrhodamine isothiocyanate for actin visualization. In the upper panel, the EC were incubated for 20 h with medium or BS-I and analyzed immediately or for 20 h with BS-I and analyzed after additional 24 h in medium alone. In the lower panel, the EC were incubated for 48 h with medium or 4 h with BS-I and examined immediately or for 4 h with BS-I and examined after additional 48 h in medium alone.

EC resistance to C is temporally separated from the proinflammatory response that follows lectin stimulation and requires protein synthesis

It was previously shown that, after a few hours of incubation, BS-I induces activation of EC, including the expression of E-selectin (28). Therefore, we compared the expression of E-selectin with resistance to C immediately and at 1 h and 16 h following stimulation with BS-I for 4 h (Fig. 12⇓). Resistance to C did not occur immediately or at 1 h after lectin exposure but was pronounced at 16 h. In contrast, E-selectin expression was maximal immediately or 1 h after lectin treatment, but was markedly reduced at 16 h (Fig. 12⇓). Thus, there is a clear temporal separation between these two manifestations of EC activation. We then investigated whether protein synthesis is required to induce resistance to C by incubating EC with BS-I in the presence of the protein synthesis inhibitor cycloheximide. Cycloheximide at a low concentration (0.2 μM) during the 18 h of incubation with BS-I interfered with development of resistance (Fig. 13⇓), as did cycloheximide at a higher concentration (9 μM) added during 4 h of incubation with BS-I and followed by 16 h of incubation in medium without BS-I or cycloheximide (results not shown). These experiments suggest that protein synthesis is a requirement for induction of resistance to the MAC.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 12.

Time course of development of resistance to C-mediated killing in comparison to expression of E-selectin by EC stimulated with BS-I. EC were incubated for 4 h at 37°C with 100 μg/ml BS-I, washed, and then incubated with medium alone for the indicated time periods. After washing, the cells were either tested for susceptibility to C (left panel) or fixed and tested for E-selectin expression by ELISA (right panel). Results are representative of three experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 13.

Induction of resistance to C killing in EC by lectin requires protein synthesis. EC were preincubated at 37°C for 18 h in DMEM-5% FBS or with DMEM-FBS plus 50 μg/ml BS-I in the absence or presence of 0.2 μM cycloheximide. After washing, the cells were tested for susceptibility to C with 40% human serum for 4 h at 37°C. At the concentrations used the cycloheximide was not toxic to the EC. Results are representative of two similar experiments.