Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3

Abstract

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl⁻ secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl⁻ secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl⁻ secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl⁻ secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl⁻ secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Physiologic salt and water secretion from the intestine depends on the activation of Cl⁻ channels in the apical membrane of epithelial cells lining the crypt, or secretory gland of the intestine (1, 2). These cells, termed “undifferentiated Cl⁻ secreting crypt cells” (3), express the necessary ion channels, pumps, and cotransporters on their basolateral membranes to amass Cl⁻ ions inside the cell (3). Activation of Cl⁻ channels in the apical membrane allows Cl⁻ to flow out of the cell down its electrochemical gradient into the crypt lumen. This ion flux hyperpolarizes the crypt lumen that drives Na⁺ and water across interepithelial tight junctions to produce a secretory response. Movement of Cl⁻ out of the cell through apical Cl⁻ channels is rate limiting in the secretory response. As such, intestinal fluid secretion is subject to regulation by neurotransmitter-, signaling peptide-, and/or nucleotide-dependent mechanisms that activate Cl⁻ channels in the apical membrane of the undifferentiated crypt cell (4).

Paneth cells are located in the small intestine at the crypt base adjacent to the undifferentiated Cl⁻ secreting cells (5). Paneth cells have been implicated in mucosal host defense because they constitutively and inducibly secrete lysozyme and defensins (5). Neutrophil defensins are cationic, amphiphilic, cyclic peptides that have the capability to insert into artificial phospholipid bilayers and cell membranes to form anion conductive pores (6, 7). This activity may explain the cytotoxic effect of neutrophil defensins on prokaryotic and eukaryotic cells in vitro (8). Because intestinal defensins or cryptdins are homologous in structure and function to defensins isolated from mammalian phagocytic cells (5, 9, 10), and undifferentiated Cl⁻ secreting crypt cells are exposed to enteric defensins discharged from Paneth cells in vivo, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of Cl⁻ secreting epithelial cells in culture (11). We found that two mouse intestinal defensins, cryptdins 2 and 3, can selectively permeabilize apical cell membranes of the human intestinal cell line T84 to elicit a physiologic secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues and identify a previously undescribed mechanism of paracrine signaling that in vivo may involve the transfer of channel-forming peptides from granules of one cell type to the apical membrane of its neighbor.

MATERIALS AND METHODS

Cell Culture, Electrophysiology, and Cyclic Nucleotide Assay.

Human intestinal T84 cells obtained from American Type Culture Collection were cultured and passaged as described (11). When grown on permeable supports, T84 cells form confluent monolayers of columnar epithelia that display polarized apical and basolateral membranes, high transepithelial resistances, and a regulated Cl⁻ secretory pathway analogous to that found in native crypt epithelium (11). Cl⁻ secretion was assessed as a short circuit current (Isc) using standard electrophysiologic techniques (12). cAMP and cGMP were assessed in ethanol extracts of T84 cell monolayers by radioimmune assay kit (NEN). Hanks’ balanced salt solution (HBSS; containing 1.67 mM CaCl₂/0.8 mM MgSO₄/5 mM KCl/0.45 mM KH₂PO₄/137 mM NaCl/0.33 mM Na₂HPO₄/5 mM glucose/10 mM Hepes, pH 7.4) was used for all assays unless otherwise stated.

Cryptdin Purification and Synthesis.

A low molecular weight peptide fraction (P-60 cryptdin fraction) from which all known cryptdins have been purified was prepared by Biogel P-60 gel chromatography of an acid extract homogenate of adult outbred Swiss Webster mouse small intestine (5). Further purification by reversed-phase HPLC yielded a cryptdin-enriched pool (HPLC cryptdin pool) that contained mouse cryptdins 1–6 as well as a number of other noncryptdin proteins (5). Mouse cryptdins 1–6 were purified to homogeneity from this fraction by HPLC (5, 9). In addition, some studies were carried out using synthetic, folded, and oxidized cryptdin 3, prepared as described for cryptdin 1 (5). Synthetic and natural cryptdin 3 peptides were shown to have identical physicochemical and antimicrobial characteristics (ref. 13, D. Tran and M.E.S., unpublished data).

Cryptdin-Induced Pore Formation.

Nonpolarized T84 cells grown on glass coverslips or polarized monolayers grown on filter supports were incubated at 37°C for 30 min in HBSS containing the membrane impermeant fluorophore 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-acid, 0.1 μM) with or without the addition of cryptdin 3 (100–600 μg/ml). At the end of the exposure, coverslips or monolayers on their filter supports were washed in fresh HBSS containing 0.1 μM BCECF at 37°C to remove the peptide. After an additional 10 min, coverslips or monolayers were washed again in fresh HBSS and examined by epifluorescence (490 nM excitation, 520 emission) and bright field microscopy using Nomarski optics.

RESULTS

An initial indication that cryptdins may induce chloride secretion was observed in experiments using the P-60 cryptdin fraction (see Materials and Methods) that is known to contain mouse cryptdins 1–6. When applied to apical membranes of T84 cells, the peptide fraction elicited an increase in transepithelial short circuit current (Isc, Fig. 1A) without a concomitant rise in intracellular cGMP or cAMP (Fig. 1 B and C). These data indicated that the secretory response was not due to the known apically acting secretory agonists guanylin (14) or Escherichia coli heat stable toxin (15), because these peptides elicit a secretory response through cGMP-mediated signal transduction. In addition, the adenosine-receptor inhibitor 8-phenyltheophyline had no detectable effect on the Isc response elicited by components of the HPLC cryptdin pool (see Materials and Methods, and Fig. 1 D and E), indicating that the Isc was independent of apically acting adenosine (16). As guanylin, E. coli heat stable toxin, and adenosine are the only known compounds that elicit a secretory response by interacting with receptors on the apical membrane, these results suggested the presence of a novel secretory agonist in the cryptdin-enriched fraction.

Figure 1.

Cryptdin-enriched fractions of mouse intestinal extract elicit a Cl⁻ secretory response from human intestinal T84 cell monolayers. (A) Time course of Cl⁻ secretion elicited by peptide fraction (50 μg/ml in HBSS, ▪), or vehicle alone (0.01% acetic acid, □) applied to apical membranes of T84 cell monolayers grown on permeable supports. The cAMP agonist vasoactive intestinal peptide (VIP, added at 52 min) elicited a secretory response from control monolayers exposed to vehicle alone. (B and C) cAMP and cGMP responses to peptide fraction. Monolayers exposed to vehicle alone (basal) or to the cGMP-agonist guanylin or cAMP-agonist forskolin provide two point calibration. (D) Time course of Cl⁻ secretion elicited by vehicle alone (buffer) or using the HPLC cryptdin pool (see Materials and Methods) in the presence or absence of the adenosine inhibitor 8-phenyltheophyline (1 μM). (E) Time course of Cl⁻ secretion elicited by adenosine (10 μM) in the presence or absence of 8-phenyltheophyline.

To determine whether cryptdins were directly responsible for the observed secretory response, we examined Cl⁻ secretion induced by assaying individually the activities of cryptdins 1–6 purified from mouse small intestine (5, 9, 10). At concentrations of 40 μg/ml, cryptdins 2 and 3 elicited a Isc response that could be reversed completely by removing the peptides from the apical reservoir (Fig. 2A, “Wash”).

Figure 2.

Cryptdins 2 and 3 elicit a reversible Cl⁻ secretory response from human intestinal T84 cell monolayers. (A) Time course of Cl⁻ secretion elicited by purified murine cryptdins 1–6. Cryptdin 1 (20 μg/ml) or cryptdins 2–6 (40 μg/ml) in HBSS were applied to apical membranes of T84 cell monolayers at 37°C. At 120 min, cryptdins were removed from the apical reservoir by washout in >100 volumes. of HBSS containing 0.1% BSA. In separate experiments, cryptdini at 40 μg/ml was also inactive. (B) Dose dependency of Cl⁻ secretion for cryptdin 3. Data represent peak currents 30 min after apical administration of cryptdin 3 (circles and squares represent two independent experiments). Data were fit to Michaelis Menten model (R² = 0.98). (C) Effect of removing Cl⁻ ions from transport buffers on the secretory response elicited by cryptdin 3 or vehicle alone (0.01% acetic acid). T84 cells were preincubated in Cl⁻ free buffer or HBSS and exposed to cryptdin 3 (100 μg/ml) or vehicle alone. At 42 min, Cl⁻ was added back (110 mM final) to Cl⁻ free buffers. At 57 min (∗), the cAMP-agonist vasoactive intestinal peptide (5 nM) was applied to control monolayers not treated with cryptdin 3 but incubated sequentially with Cl⁻-free and then Cl⁻ replete buffers as described above. Cl-free buffer consisted of 140 mM Na⁺ gluconate, 5 mM K⁺ gluconate, 1.25 mM Ca²⁺ acetate, 1 mM Mg²⁺ gluconate, 5 mM KH₂PO₄/Na₂HPO₄, 20 mM Hepes, and 5 mM glucose (pH 7.4).

The effect of cryptdin 3 on T84 cell short circuit current was dose-dependent. When applied to apical membranes, cryptdin 3 elicited an increase in Isc with maximal currents of 65 μA/cm² at 600 μg/ml and an apparent EC₅₀ of 250 μg/ml (Fig. 2B). Such concentrations are likely to be achieved within the crypt in vivo given the small volume of the long but narrow crypt lumen (≈5 μm × 250 μm) (3), and the high apparent concentration of cryptdins in Paneth cell secretory granules that approximate concentrations of defensins in phagolysosomes of neutrophils (1–10 mg/ml, refs. 17 and 18). At concentrations below 300 μg/ml (incubated for 30 min at 37°C), the Isc responses to cryptdin 3 were fully reversible. At higher concentrations, however, 30 min incubations with cryptdin 3 resulted in a gradual reduction of Isc (data not shown) that paralleled a progressive and eventual complete loss of monolayer resistance, perhaps due to cytotoxicity (19).

The source of the Isc induced by cryptdin 3 was identified as a Cl⁻ current, the primary transport process responsible for the secretory response across mucosal surfaces. Substitution of membrane impermeant gluconate for Cl⁻ in both apical and basolateral transport buffers abrogated completely the Isc induced by cryptdin 3, due to depletion of intracellular Cl⁻ (Fig. 2C, ⋄) The dependency on Cl⁻ was confirmed by restoration of the Isc response after replenishing the basolateral reservoir with Cl⁻ at 43 min. Replenishment was specific for cryptdin 3-treated monolayers, because adding Cl⁻ back to monolayers not exposed to cryptdin 3 had no effect on short circuit currents (□). The viability of control monolayers was demonstrated by the brisk secretory response to vasoactive intestinal peptide (5 nM) added at 57 min. These data show that the Isc induced by cryptdin 3 was Cl⁻ dependent. Further evidence that the cryptdin 3 induced Isc’s represent Cl⁻ transport was provided by the use of bumetanide (10 μM), a specific inhibitor of the Na/K/2Cl uptake pathway. Na/K/2Cl cotransport is primarily responsible for Cl⁻ uptake in secretory epithelia, and inhibition of this cotransporter blocks secondary active accumulation of Cl⁻ by epithelial cells. In agreement with data obtained by ion substitution (Fig. 2C), we found that bumetanide reduced cryptdin-3-induced Iscs by 75%. Taken together, these data show that cryptdin 3 elicits a Cl⁻ secretory response from intestinal T84 cells. Such a response could occur by activation of pre-existing apically located Cl⁻ channels or by formation of new anion-conductive pores resulting from the insertion of cryptdin 3 into the apical membrane.

To obtain direct evidence that the cryptdin-induced secretory response in T84 cells may be due to formation of cryptdin-based channels, we examined the effect of cryptdin 3 on the permeability of T84 cell plasma membranes to the impermeant fluorophore BCECF-acid (450 Da). Fig. 3A shows that cryptdin 3 markedly increased membrane permeability of nonpolarized T84 cells to this organic acid. Apical membranes of polarized cells were also permeabilized by cryptdin 3 (Fig. 3B), but the amount of BCECF-acid uptake into individual cells was not uniform. This was not dependent on cryptdin 3 concentration (200–600 μg/ml), and likely represents heterogeneity of cell phenotype in the T84 cell clone (11). Further evidence that cryptdins can permeabilize selectively apical membranes of polarized cells are provided by studies that show that cryptdins 2 and 3 induce Cl⁻-dependent transepithelial currents (Figs. 1 and 2), and that cryptdin 3 will synergize with the muscarinic agonist carbacol to elicit a Isc response (Fig. 3C). Muscarinic agonists elicit a secretory response by activating basolateral K⁺ conductances that hyperpolarize the cell and drive Cl⁻ efflux through channels (in this case, cryptdin-induced pores) located on the contralateral apical membrane (20). Defensins purified from rabbit phagocytic cells (21) and kidney (22) also elicit ion flux from intestinal epithelial cells. This response, however, occurs at nanomolar concentrations, and exhibits dependency on extracellular Ca²⁺. This activity appears to be distinct from the channel-forming action displayed by cryptdin 3 on T84 cell membranes.

Figure 3.

Cryptdin 3 forms channels in T84 cell membranes. (A) Cryptdin 3 (280 μg/ml) permeabilizes T84 cells to the membrane impermeant fluorophore BCECF-acid. Photographs were taken at 1 and 10 sec exposures. (Bar = 11 μm.) (B) Cryptdin 3 (200 μg/ml) permeabilizes apical membranes of T84 cell clusters to BCECF-acid. Photographs were taken at 1, 5, and 10 sec exposures. Data are representative of four independent experiments. Magnification as in A. (C) Synergistic effect of cryptdin 3 with the muscarinic agonist carbacol. T84 cell monolayers were treated apically with cryptdin 3 (100 μg/ml) or vehicle alone. At 25 min, monolayers were exposed basolaterally to the muscarinic agonist carbacol (100 μM). (D) Diagram of the intestinal crypt. Cryptdin secreting Paneth cells are located at the base of the small intestinal crypt, and consequently cryptdins are exposed to the apical membrane of Cl⁻ secretory cells that line the remainder of the gland.

DISCUSSION

The results of these studies show that cryptdins 2 and 3 induce a physiologic Cl⁻ secretory response in human intestinal T84 cells. Cryptdin 3 appears to act by forming anion conductive channels in the apical (lumenal) membrane. The formation of anion conductive pores in model membranes has been demonstrated for the homologous human neutrophil defensins applied to planar and vesicular lipid bilayers (7, 23). Multimeric defensin pores have estimated diameters of ≈20 Å that permit the transit of 4,000-Da dextrans (23). Formation of cryptdin-containing channels in apical membranes of polarized T84 cells results in a concentration-dependent and reversible Cl⁻ secretory response, suggesting that cryptdins may act as novel paracrine regulators of crypt secretory cell function in vivo (Fig. 3D).

The secretory activities elicited by interaction between cryptdins and T84 cells were highly specific for individual peptides, as cryptdin 1 and cryptdins 4–6 were inactive under these in vitro conditions. These data identify specific residues of the cryptdin molecule that appear to confer bioactivity. Cryptdins 1, 2, 3, and 6 differ only at residue positions 10, 15, 29, and 31 (Fig. 4) (10). The two biologically active peptides, cryptdins 2 and 3, contain arginine at position 15 while cryptdins 1 and 6 contain glycine side chains at that position, and they are inactive. Thus, Arg-15 in cryptdins 2 and 3 may be important for channel formation in human T84 cells. As cryptdins 2 and 3, but not cryptdins 1 and 6, display antimicrobial activity against trophozoites of Giardia lamblia (24), perhaps the bulky, cationic arginine on the surface turn at position 15 facilitates interaction of enteric defensins with eukaryotic membranes. The present data also show that the secretory response elicited by cryptdin 3 was more extensive than that induced by cryptdin 2. Because cryptdins 2 and 3 differ only at residue position 10 (threonine vs. lysine, respectively), residues at position 10 may also be important in cryptdin action on T84 cells. By analogy with the known crystal or solution structures of human neutrophil defensins HNP-1 and -3 and rabbit defensins NP-1 and-5, the amino acids at position 10 are predicted to be involved in a conserved turn on the peptide surface and, we hypothesize, are positioned to influence the interaction between cryptdin and cell membranes.

Figure 4.

Amino acid sequences of cryptdins 1–6. Primary structures are shown in single letter amino acid code, and aligned to maximize sequence similarity with hyphens denoting gaps in the cryptdin 4 sequence. ∗, Amino acid positions where cryptdins 1, 2, 3, and 6 differ. By analogy with the neutrophil defensins HNP-1, HNP-3, NP-2, and NP-5, amino acids at positions 10 and 15 are predicted to be located at conserved turns on the surface of the molecule (10). Variations at these positions have been shown to confer effects on the potency of antimicrobial activities (10, 24).

The six cryptdins tested are present at different relative abundance in vivo, and the relative content of individual cryptdins in different intestinal crypts is unknown (5, 10). In particular, cryptdin 3, the most potent peptide in eliciting a secretory response from T84 cells, represents one of the least abundant defensins purified from full length mouse intestine. Based on peptide recoveries, cryptdins 1, 2, and 6 are judged to be most abundant and occur at approximately equivalent levels. As cryptdin 2 also displays activity in eliciting a secretory response from T84 cells, it may be the more relevant biologically-active peptide responsible for paracrine signaling in vivo. Also, as synergy between different neutrophil defensins has been described for antimicrobial activity (25), and the crystal structure of HNP-3 is a noncovalent dimer (26), cryptdin isoforms discharged from Paneth cells may form intermolecular associations that could augment their activity in the intestinal crypt. Such potential associations may occur between different cryptdins, or between cryptdins and additional proteins or peptides released by Paneth cells, or both. Finally, Paneth cells also contain mRNA encoding guanylin (27), a peptide that elicits intestinal Cl⁻ secretion by paracrine signaling through classical receptor-mediated cGMP-dependent pathways (14). These findings suggest that Paneth cells may play a previously unrecognized central role in regulating intestinal fluid secretion by discharging cryptdins, guanylin, or both into the crypt lumen.

In summary, these studies show that cryptdins 2 and 3 are capable of inducing a Cl⁻ secretory response by forming anion conductive channels in the apical membranes of human intestinal T84 cells in culture. Thus, in vivo, after discharge from Paneth cells at the base of the crypt, cryptdins may interact with and insert into apical membranes of adjacent epithelial cells that are electrochemically poised to secrete Cl⁻ (Fig. 3D). If such channel formation occurs in the intestinal crypt, it would lead to salt and water secretion, causing the crypt lumen to be flushed after Paneth cell discharge. Because cryptdin-based channel formation is dependent on peptide concentration (this study and refs. 6 and 7), the act of flushing the crypt lumen would reverse the cryptdin-induced secretory response by diluting the peptides to levels below the effective concentration. This balance between luminal cryptdin concentrations sufficient to induce Cl⁻ secretion and levels below the effective range may serve to protect crypt epithelial cells from possible cytotoxic effects while delivering these antimicrobial peptides to cell surfaces higher in the crypt or within the gut lumen itself.

ABBREVIATIONS

HBSS

Hanks’ balanced salt solution

Isc

short circuit current

BCECF-acid

2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein