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Originally published online as doi:10.1189/jlb.1005566 on July 20, 2006

Published online before print July 20, 2006
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(Journal of Leukocyte Biology. 2006;80:492-499.)
© 2006 by Society for Leukocyte Biology

Mucosal IL-8 and TGF-ß recruit blood monocytes: evidence for cross-talk between the lamina propria stroma and myeloid cells

Lesley E. Smythies*,1, Akhil Maheshwari{dagger}, Ronald Clements{ddagger}, Devin Eckhoff{ddagger}, Lea Novak§, Huong L. Vu, L. Meg Mosteller-Barnum*, Marty Sellers* and Phillip D. Smith*,||

* Departments of Medicine (Gastroenterology),
{dagger} Pediatrics (Neonatology),
{ddagger} Surgery (Gastrointestinal and Transplantation),
§ Pathology, and
Microbiology, University of Alabama at Birmingham; and
|| VA Medical Center, Birmingham, Alabama

1 Correspondence: Department of Medicine (Gastroenterology), UAB (ZRB 633), 703 19th St. South, Birmingham, AL 35294. E-mail: lesmy{at}uab.edu

ABSTRACT

The lamina propria of the gastrointestinal mucosa contains the largest population of mononuclear phagocytes in the body, yet little is known about the cellular mechanisms that regulate mononuclear cell recruitment to noninflamed and inflamed intestinal mucosa. Here, we show that intestinal macrophages do not proliferate. We also show that a substantial proportion of intestinal macrophages express chemokine receptors for interleukin (IL)-8 and transforming growth factor-ß (TGF-ß), and a smaller proportion expresses receptors for N-formylmethionyl-leucyl-phenylalanine and C5a, but, surprisingly, they do not migrate to the corresponding ligands. In contrast, autologous blood monocytes, which express the same receptors, do migrate to the ligands. Blood monocytes also migrate to conditioned medium (CM) derived from lamina propria extracellular matrix, which we show contains IL-8 and TGF-ß that are produced by epithelial cells and lamina propria mast cells. This migration is specific to IL-8 and TGF-ß, as preincubation of the stroma-CM with antibodies to IL-8 and TGF-ß significantly blocked monocyte chemotaxis to the stromal products. Together, these findings indicate that blood monocytes are the exclusive source of macrophages in the intestinal mucosa and underscore the central role of newly recruited blood monocytes in maintaining the macrophage population in noninflamed mucosa and in serving as the exclusive source of macrophages in inflamed mucosa.

Key Words: intestinal macrophage • chemotaxis • migration • recruitment • inflammation

INTRODUCTION

Intestinal macrophages are an important host-defense cell in noninflamed and inflamed intestinal mucosa. In noninflamed mucosa, recruited mononuclear phagocytes are thought to replace senescent resident macrophages and, as we recently showed [1 ], acquire profound inflammatory anergy in the process. This inflammatory anergy prevents resident macrophages from participating in the inflammatory cascade and thereby promotes the absence of inflammation characteristic of normal intestinal mucosa [2 ]. In contrast, intestinal inflammation is characterized by the accumulation of large numbers of mononuclear phagocytes, which produce a wide array of potent, soluble mediators, including cytokines and chemokines [3 ]. These soluble mediators play an important role in the pathogenesis of intestinal, inflammatory processes such as Crohn’s disease [4 5 6 7 ]. Understanding the cellular mechanisms that regulate mononuclear cell recruitment to noninflamed and inflamed intestinal mucosa has received little investigative attention. Indeed, the local chemokine ligands that recruit mononuclear phagocytes to the mucosa and the source of the recruited cells—blood versus the mucosa itself—are not known.

Cell recruitment, referred to as chemotaxis, is a highly regulated, receptor-mediated process in which cells migrate in a concentration-dependent manner to chemokines, bacterial components, and complement factors [8 ]. In the past, investigating mucosal cell chemotaxis has been limited by difficulties in isolating and culturing mucosal cells, particularly macrophages. Recently, we overcame these difficulties and developed a technique to isolate and purify primary human macrophages from normal jejunum [9 , 10 ]. Using these cells, together with autologous, purified blood monocytes, we investigated the biological parameters of mononuclear cell recruitment to the mucosa. Here, we show that intestinal macrophages express chemokine receptors, but, surprisingly, they do not migrate to chemoattractant ligands, whereas autologous blood monocytes, which express the same receptors, migrate to the ligands and to chemokines released by lamina propria extracellular matrix (ECM). These findings underscore the central role of newly recruited blood monocytes in maintaining the macrophage population in noninflamed mucosa and in serving as the exclusive source of macrophages in inflamed mucosa. Piecing together the cellular and molecular pathways involved in mononuclear cell recruitment to the mucosa will provide critical information for developing novel strategies to blunt, modulate, or inhibit mucosal inflammation in intestinal diseases, including inflammatory bowel disease and infectious gastroenteritis.

MATERIALS AND METHODS

Intestinal macrophages, intestinal epithelial cells, and blood monocytes
Intestinal macrophages were isolated from enzyme-digested, intestinal tissue sections and purified by counterflow centrifugal elutriation, as we have described in detail [9 , 10 ]. Briefly, sections of normal human jejunum obtained with Institutional Review Board approval from subjects undergoing elective gastrojejunostomy for obesity or healthy organ transplantation donors were dissected into mucosa and submucosa. The mucosa was rinsed in Ca++- and Mg++-free phosphate-buffered saline (PBS) and washed in Hanks’ balanced saline solution (HBSS) plus dithiothreitol (200 µg/ml) to remove residual mucus and then in HBSS containing 0.2 M EDTA plus 10 mM 2-mercaptoethanol to remove the epithelium. Epithelial cells were separated from the intraepithelial lymphocytes and then purified, as we have described previously in detail [11 ]. The tissue sections devoid of epithelium next were minced and treated with neutral protease Dispase, 75 µg/ml (Grade I, specific activity >6 U/mg with <0.01 ng/ml endotoxin by Limulus amebocyte lysate assay, Cambrex, Walkersville, MD), to release the lamina propria mononuclear cells. Macrophages were purified from the released mononuclear cells by gradient sedimentation followed by elutriation [9 , 10 , 12 ]. The cells isolated by this procedure were routinely >98% viable by propidium iodide (PI) staining, and morphologic and ultrastructural analysis confirmed the cells were macrophages [1 , 9 , 10 , 13 ]. The absence of detectable CD3, CD20, CD69, CD34, CD83, and CD103 on the purified cell populations confirmed the absence of contaminating T or B lymphocytes, natural killer cells, dendritic cells, and the consistently high purity (>98%) of the intestinal macrophages [1 , 9 , 13 ]. Blood monocytes from the tissue donors were purified by positive sorting with magnetic beads (MACS MicroBeads, Miltenyi Biotec, Auburn, CA) conjugated with anti-CD14 monoclonal antibodies (mAb; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. The purified monocytes were treated with Dispase according to the macrophage isolation protocol so that intestinal macrophages and blood monocytes were exposed similarly to neutral protease enzyme. Blood monocytes displayed the same purity and viability as the intestinal macrophages.

Stroma-conditioned media (S-CM)
After the mononuclear cells were removed from the lamina propria stroma (ECM), as described above, the cell-free stroma was cultured in RPMI for 24 h (1 g wet weight stromal tissue/ml) [1 ]. The culture supernatant was harvested, sterile-filtered (0.2 mm syringe filter, Corning Inc., NY), and frozen at –70°C. In preliminary studies, culture supernatant, referred to as S-CM, did not alter blood monocyte or intestinal macrophage viability (90%) for up to 4 days, as assessed by PI uptake. Endotoxin, protein, and protease content of each S-CM were determined by commercially available enzyme-linked immunosorbent assays (ELISAs).

Chemotaxis assay
Intestinal macrophage and blood monocyte chemotaxis were assessed in microchemotaxis chambers [14 , 15 ] using a variation of our chemotaxis assay described previously [16 , 17 ]. Briefly, monocytes were stained with the fluorescence dye Calcein AM (2 µM; Molecular Probes, Eugene, OR) [14 ], and intestinal macrophages were stained similarly but with a predetermined optimal concentration of 4 µM Calcein AM. Next, varying concentrations of recombinant interleukin (rIL)-8 (R&D Systems), recombinant transforming growth factor-ß (rTGF-ß; R&D Systems), N-formylmethionyl-leucyl-phenylalanine (fMLP; Sigma Chemical Co., St. Louis, MO), C5a (Sigma Chemical Co.), or S-CM in 300 µL HBSS with 0.1% bovine serum albumin (BSA) were placed in triplicate in lower wells of 96-well microchemotaxis chambers (ChemoTx System, Neuro Probe, Gaithersburg, MD). After a polycarbonate filter membrane (5 µm pores; Neuro Probe) was positioned between the lower and upper wells, 80,000 intestinal macrophages or blood monocytes (72 µl 1.1x106 cells/mL) with similar viability (98%) in HBSS plus 0.1% BSA were placed in the upper well. Control wells contained 0–80,000 calcein-stained monocytes or intestinal macrophages per well to generate their respective standard curves. The microchemotaxis chamber plate was incubated 60 min at 37°C in 5% CO2 in humidified air. After incubation, nonmigrated cells on the top of the filter were removed with a smooth-edged wiper, and the plate was centrifuged at 200 g for 5 min to collect migrated cells attached to the underside of the filter, which was removed, and the optical density for each well was determined in an ELISA reader (excitation 485 nm, emission 530 nm). The number of cells that migrated from the upper well into the lower well was determined by comparing the mean calcein fluorescence signal of the cells in test wells with that of the standard curve generated from known numbers of fluorescence-labeled cells.

Flow cytometric analysis
Fresh intestinal macrophages and blood monocytes (2x105) were incubated, first, in 10% human AB serum for 30 min and then in optimal concentrations of fluorescein isothiocyanate (FITC)-labeled mAb to IL-8 receptor [IL-8R; CXC chemokine receptor 1 (CXCR1) and CXCR2], TGF-ß receptor I (TGF-ßRI) and RII, fMLP receptor (fMLPR), and C5a receptor (C5aR; R&D Systems) or control FITC-labeled, irrelevant antibody of the same isotype. Blood monocytes were also analyzed for CD13 (R&D Systems), a pan-myeloid cell marker absent on T cells, CD14 (R&D Systems), and the lipopolysaccharide (LPS) receptor, before and after migration to S-CM. After staining, the cells were washed, fixed in 1% parafomaldehyde, and analyzed by flow cytometry. Data were analyzed with CellQuest software (Becton Dickinson, San Jose, CA).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
RNA was isolated from fresh intestinal macrophages and homologous blood monocytes. After RT, the resultant cDNA (5 µl) was amplified in a 50-µl reaction containing 0.25 µl Taq polymerase (5 U/ml; Gene Amp PCR system, Perkin Elmer Cetus, Norwalk, CT), 4 µl deoxy-unspecified nucleoside 5'-triphosphates (2.5 mmol/l; Promega, Madison, WI), 2 µl MgCl2 (25 mmol/l), 5 µl 10x PCR reaction buffer, and 2.5 µl primers for TGF-ßRI and RII [18 ] (denaturation: 94°C for 1 min; annealing: 57°C for 1 min; extension: 72°C for 2 min, 35 cycles), CXCR1 and R2 (R&D Systems; denaturation: 94°C for 45 s; annealing: 55°C for 45 s; extension: 72°C for 45 s, 35 cycles), C5aR [19 ] (denaturation: 95°C for 30 s; annealing: 60°C for 30 s; extension: 68°C for 2 min, 40 cycles), fMLPR [20 ] (denaturation: 96°C for 15 s; annealing: 55°C for 30 s; extension: 72°C for 3 min, 35 cycles), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; denaturation: 95°C for 1 min; annealing: 60°C for 2 min; extension: 72°C for 3 min, 25 cycles) [13 ], and the products were detected by ethidium bromide-stained agarose gels. In separate RT-PCR analyses, purified jejunal epithelial cells were analyzed by RT-PCR for IL-8, TGF-ß, tumor necrosis factor {alpha} (TNF-{alpha}), and ß-actin using our protocol and primer sequences described previously [21 ], before and after a 4-h exposure to Helicobacter pylori urease [22 ], a prominent immunodominant antigen.

Cell proliferation analysis
Intestinal macrophage and blood monocyte proliferation were assessed by autoradiographic evaluation of 3H-thymidine ([3H]TdR) incorporation. Freshly isolated intestinal macrophages, blood monocytes, and foreskin fibroblasts (1x106 cells/0.5 mL/well) were plated in 24-well tissue-culture plates in RPMI supplemented with antibiotics and 10% human AB serum and then pulsed with 5 µL [3H]TdR, 10 µCi/mL (specific activity 20 Ci/mmol; New England Nuclear, Boston, MA), between 0 and 24 h or 24 and 48 h (to allow incorporation into already-proliferating cells). At the end of each pulse, the cells were cytospun onto silanized glass slides (two slides per well), air-dried, and fixed in 1.5% glutaraldehyde. The experiment also was performed in the presence of human recombinant granulocyte macrophage-colony stimulating factor (rGM-CSF), 500 units/mL, under the same conditions. The slides were examined by autoradiography, and the number of positive cells among 400–600 cells in 10 fields per slide was determined. The labeling index indicates the number of positive cells divided by the total number of cells counted.

Chemokine levels
The amounts of IL-8 and TGF-ß in the S-CMs were determined by ELISA (R&D Systems); the TGF-ß determinations were made after acid activation of the S-CM.

Immunohistochemical analysis
Normal human jejunum was embedded in optical cutting temperature compound, snap-frozen, and stored at –20°C. Frozen sections (5 µm) were fixed in 3.7% paraformaldehyde/PBS for 30 min, washed in PBS, and blocked with 3% BSA for 30 min, after which mouse anti-human mAb to IL-8 and TGF-ß (both R&D Systems) and an isotype-matched control antibody diluted 1:50 in BSA were applied separately to the sections for 2 h at room temperature. After washing with PBS, fluorescence-labeled (Alexa Fluor 488 for green and Alexa 568 for red, Invitrogen Molecular Probes, Eugene, OR) rabbit anti-mouse immunoglobulin G (IgG), diluted 1:100 in BSA, was applied for 2 h at room temperature, protected against light exposure. Cell nuclei were stained with Hoechst 33342 antibody (Calbiochem, San Diego, CA), diluted 1:1000 in PBS, applied for 3 min, followed by PBS wash. Imaging was performed using laser-scanning confocal microscopy equipped with ultraviolet, argon, and krypton lasers.

RESULTS

Intestinal macrophages and blood monocytes do not proliferate
To determine whether cell proliferation could contribute to the macrophage populations in noninflamed and inflamed mucosa, we first determined whether intestinal macrophages and blood monocytes could incorporate [3H]TdR as a measure of cell division. As shown in Table 1 , cultured fibroblasts, but not intestinal macrophages or blood monocytes, incorporated [3H]TdR spontaneously into their nuclei. The addition of optimal concentrations of GM-CSF to the cultures did not induce incorporation of [3H]TdR by intestinal macrophage or blood monocyte nuclei (data not shown). These results indicate that under the conditions of our assay system, fibroblasts, not mononuclear phagocytes, are capable of cell division.


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  		Table 1. Analysis of [3H]TdR Incorporation by Intestinal Macrophages and Blood Monocytes

 
Intestinal macrophages express chemokine receptors
The inability of mononuclear phagocytes to proliferate suggested that recruited macrophages and/or blood monocytes are the source of mucosal macrophages in the small intestine. Therefore, we next analyzed intestinal macrophages and blood monocytes for key receptors involved in cell recruitment, including the IL-8Rs CXCR1 and CXCR2, the TGF-ßRs TGF-ßRI and RII, and receptors for the synthetic bacterial chemotactic peptide fMLP [23 , 24 ] and the inflammatory ligand C5a [19 , 25 ]. Similar to autologous blood monocytes, substantial numbers of intestinal macrophages from each of five donors expressed CXCR1 and CXCR2, as well as TGF-ßRI and RII (Fig. 1 ). In contrast, low numbers of intestinal macrophages expressed fMLPR and C5aR (Fig. 1) . Although receptors for fMLP and C5a were absent on intestinal macrophages from three of the five donors, intestinal macrophages consistently expressed mRNA for both of these receptors (n=5; Fig. 2 ) and for CXCR1, CXCR2, TGF-ßRI, and TGF-ßRII (data not shown).


Figure 1
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  		Figure 1. Intestinal macrophage and blood monocyte expression of receptors for IL-8 (CXCR1 and CXCR2), TGF-ß (TGF-ßRI and TGF-ßRII), fMLP (fMLPR), and C5a (C5aR). Matched jejunal tissue and blood cells were prepared and stained with optimal concentrations of receptor-specific antibodies as indicated (thick, solid line) or isotype-matched, control antibodies (thin, solid line), and ungated populations were analyzed by flow cytometry as described in Materials and Methods. Percents correspond to the number of cells that stained for the receptor above background in a representative donor (n=5).

 

Figure 2
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  		Figure 2. C5aR and fMLPR mRNA expression in lamina propria intestinal macrophages. Fresh intestinal macrophges were purified from four donors and analyzed by RT-PCR for C5aR, fMLPR, and GAPDH (control) mRNA transcripts. Products were visualized on a 2% agarose gel stained with ethidium bromide. MW, Molecular weight.

 
Intestinal macrophages do not chemotax to chemoattractant ligands
Having shown that resident intestinal macrophages express protein and message for several chemokine receptors, we next evaluated intestinal macrophages for chemotactic activity to the corresponding ligand. Surprisingly, intestinal macrophages did not chemotax to IL-8 or TGF-ß over wide concentration ranges of each ligand (Fig. 3 ), despite the presence of IL-8R and TGF-ßR on a substantial proportion of the cells (Fig. 1) . Similarly, intestinal macrophages did not migrate to fMLP or C5a, although low numbers of the cells expressed fMLPR and C5aR. In sharp contrast, blood monocytes from the same donor displayed strong, dose-dependent chemotactic activity to each of the four chemotactic ligands. These findings indicate that resident intestinal macrophages do not chemotax to important chemoattractant ligands, despite the presence of the corresponding receptor, implicating a defect in cell locomotion or chemokine receptor-mediated signal transduction in the inability of intestinal macrophages to chemotax.


Figure 3
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  		Figure 3. Chemotactic activity of intestinal macrophages and blood monocytes for IL-8, TGF-ß, fMLP, and C5a over a range of concentrations. Values represent the mean (±SEM) number of cells that migrated in triplicate wells (n=7). Insets show calcein staining of (A) monocytes and (B) intestinal macrophages.

 
Blood monocytes chemotax to stroma-derived IL-8 and TGF-ß
The ability of blood monocytes but not intestinal macrophages to migrate to inflammatory chemoattractants suggested that blood monocytes are the exclusive source of macrophages in inflamed intestinal mucosa. To investigate whether blood monocytes are also the source of macrophages in noninflamed intestinal mucosa, we investigated whether lamina propria matrix products present in S-CM recruit blood monocytes. As shown in Figure 4A , S-CM from three separate donors elicited dose-dependent, migratory responses by blood monocytes in the microchemotaxis assay. Importantly, the S-CMs contained IL-8 and TGF-ß (Fig. 4A , inset) at levels that were within or approached the concentration ranges that induced chemotaxis (Fig. 3) . In general, the S-CM with the lowest level of IL-8 was the least chemotactic, whereas the S-CM with the highest level was the most chemotactic. To evaluate the chemoattractant activity of IL-8 and TGF-ß in S-CM, we preincubated S-CM with anti-IL-8 antibodies, anti-TGF-ß antibodies, or isotype-matched control antibodies and tested the S-CM for chemotactic activity. S-CM chemottractant activity was reduced >50% by preincubation with IL-8-specific antibodies and 40% after preincubation with TGF-ß-specific antibodies (Fig. 4B) but not after preincubation with control antibody (data not shown). Moreover, preincubation of the monocytes with S-CM as a source of IL-8 and TGF-ß for receptor blockade inhibited monocyte migration to S-CM by 70%. Moreover, immunofluorescence staining of jejunal sections using fluoresecence confocal microscopy showed IL-8 and TGF-ß staining in epithelium and scattered lamina propria cells (Fig. 4C) . Finally, jejunal epithelial cells in the presence or absence of stimulation with H. pylori urease express mRNA for IL-8 and TGF-ß but not TNF-{alpha} (Fig. 4D) . We have reported previously that propria mononuclear mast cells produce TGF-ß [1 ] and show here that the IL-8+ lamina propria cells stain for c-kit, a mast cell marker (Fig. 5 ), indicating that mast cells also produce IL-8. Thus, epithelial cells and mast cells are the source of the ECM (S-CM) IL-8 and TGF-ß. Analysis of jejunal tissue sections stained with isotype-matched control antibodies was negative (data not shown). These findings implicate IL-8 and TGF-ß as major monocyte chemoattractants released by the lamina propria ECM, although other, yet-to-be-identified chemokines or chemotactic ligands are likely present in the S-CM.


Figure 4
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  		Figure 4. Monocyte recruitment activity of S-CM. (A) Monocyte migration to S-CM derived from jejunal lamina propria from three separate donors (n=7). The S-CMs were prepared and normalized for protein content as described in Materials and Methods. Values represent the mean (±SEM) number of monocytes that migrated in triplicate wells. Inset shows the levels of IL-8 and TGF-ß in each undiluted S-CM. (B) Monocyte migration to S-CM before and after the S-CM was preincubated with anti-IL-8 or anti-TGF-ß antibodies (10 µg/ml) and after the monocytes were preincubated with S-CM (500 µg/mL for 1 h). Values are the mean ± SEM percent inhibition of monocyte migration to S-CM 2 in A (150 µg/ml), performed in triplicate wells (n=3). (C) Immunofluorescence microscopy for IL-8 and TGF-ß in jejunal mucosa. Serial sections of normal human jejunum were stained with anti-IL-8 or anti-TGF-ß antibodies followed by secondary green or red fluorescence-labeled antibodies to mouse IgG as described in Materials and Methods and then examined by confocal microscopy (original magnification, 200x). (D) IL-8 and TGF-ß mRNA expression in intestinal epithelial cells. Fresh and H. pylori urease-stimulated (4 h), intestinal epithelial cells were analyzed for IL-8, TNF-{alpha}, and TGF-ß mRNA by RT-PCR.

 

Figure 5
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  		Figure 5. Intestinal lamina propria cells stain for IL-8 and c-kit. Serial sections of jejunal mucosa were stained with (A) mouse anti-IL-8 antibodies followed by red fluorescence-labeled goat anti-mouse IgG antibodies and (B) rabbit anti-c-kit followed by green fluorescence-labeled goat anti-rabbit IgG antibodies and then examined by confocal microscopy. (C) Overlay of A and B shows that the IL-8+ cells were also c-kit+. Insets show a higher magnification of the cell in each panel.

 
Consistent with their profoundly down-regulated, proinflammatory function [1 ], intestinal macrophages do not express many innate response receptors such as CD14, the receptor for complexes of LPS and LPS-binding protein [1 , 9 , 10 ]. Because ~5% of blood monocytes are CD14–, we investigated whether S-CM preferentially recruited CD14– monocytes. For these experiments, we used unfractionated blood mononuclear cells, which contained CD13+ cells (monocytes) and CD13– cells (lymphocytes) as the starting population in the microchemotaxis assay. As shown in Figure 6 , left panels, the recruited population of cells contained exclusively CD13+ cells, indicating that only monocytes migrated to S-CM. The starting population also contained CD14+ cells (monocytes) and CD14– cells (lymphocytes and CD14– monocytes), but the migrated population was nearly all CD14+ cells (Fig. 6 , right panels). These findings show that S-CM did not preferentially recruit CD14– blood monocytes and suggest that the down-regulation of chemokine and chemoattractant ligand receptors on intestinal macrophages occurs after blood monocytes are recruited to the lamina propria.


Figure 6
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  		Figure 6. Phenotype of blood mononuclear cells before and after migration to S-CM. Unfractionated blood mononuclear cells were analyzed by flow cytometry for CD13 (myeloid cell marker, left panels) and CD14 (LPS receptor present on 95% of monocytes, right panels) before and after recruitment to S-CM. PE, Phycoerythrin.

 
DISCUSSION

We report the first detailed investigation into the source of macrophages in human intestinal mucosa. Blood monocytes and intestinal macrophages did not undergo cell division, indicating that mononuclear cell recruitment, not proliferation, is the likely source of mucosal macrophages. Further, blood monocytes expressed mRNA and protein for CXCR1, CXCR2, TGF-ßRI, TGF-ßRII, fMLPR, and C5aR and chemotaxed to the homologous ligands. Monocytes also chemotaxed to products released by lamina propria ECM, including IL-8 and TGF-ß. However, intestinal macrophages, which expressed mRNA and protein for IL-8R and TGF-ßR, did not chemotax to IL-8 or TGF-ß. In addition, intestinal macrophages expressed mRNA for fMLPR and C5aR, and a small proportion of the cells expressed fMLPR and C5aR, yet the macrophages did not chemotax to these ligands either. Thus, the profound inability of intestinal macrophages to chemotax, compared with the strong migratory activity of monocytes, implicates blood monocytes as the exclusive source of macrophages in intestinal mucosa.

The mucosal expression and chemoattractant activity of local TGF-ß, together with that of IL-8, indicate a mechanism for the continuous recruitment of monocytes to replace dying and senescent macrophages in noninflamed intestinal mucosa. Monocyte migration to TGF-ß was detected at the 2-pM level, establishing the sensitivity of our microchemotaxis assay and confirming TGF-ß as the most potent, natural chemotactic ligand [26 ]. Our findings also indicate that IL-8 is a strong chemoattractant for monocytes (detected at 100 pM), albeit less potent than TGF-ß. That monocytes express IL-8Rs and migrate to IL-8 confirm previous observations of IL-8 chemoattractant activity for monocytes [27 ]. The constitutive expression of IL-8 in noninflamed intestinal mucosa and the ability of stromal IL-8 to recruit monocytes underscore the in vivo relevance of monocyte recruitment by IL-8 and expand the paradigm of IL-8 as a neutrophil chemokine to a potent monocyte chemokine as well. Thus, the constitutive expression of IL-8 and TGF-ß by intestinal epithelial cells and mast cells suggests that "cross-talk" between these cells and monocytes, via the lamina propria stroma, contributes to macrophage homeostasis in the human intestinal mucosa.

The profoundly divergent abilities of blood monocytes and intestinal macrophages to chemotax to IL-8 and TGF-ß and to the inflammatory mediators fMLP and C5a implicate proinflammatory blood monocytes, not inflammation anergic intestinal macrophages, as the recruited cell to sites of inflammation in the intestinal mucosa, where they may contribute significantly to the inflammatory cytokine cascade. Indeed, increased production of TGF-ß and IL-8 reported in inflamed mucosa [28 ] likely reflects increased monocyte, not intestinal macrophage, recruitment.

As monocytes take up residence in the mucosa, they appear to lose their chemotactic function, similar to the acquired down-regulation of proinflammatory function by newly recruited monocytes, which we reported recently [1 , 13 ]. Although intestinal macrophages have been shown to emigrate from the lamina propria across damaged epithelium in an in vitro tissue model [29 ], chemotactic activity by the macrophages was not investigated. Whereas macrophages may be capable of random chemokinetic movements to enhance their phagocytosis of motile targets such as bacteria, our findings indicate that intestinal macrophages are incapable of directed movement along concentration gradients. Thus, our novel findings suggest that resident lamina propria macrophages do not contribute to the macrophage population in intestinal mucosa by cell division or recruitment. Rather, the presence of TGF-ß and IL-8 in intestinal mucosa and the ability of monocytes to migrate to stromal TGF-ß and IL-8 suggest that these cytokines/chemokines recruit blood monocytes to the lamina propria in the intestinal mucosa.

Our previous studies have shown that intestinal macrophages do not express CD14 and are profoundly down-regulated for inflammatory cytokine release, yet they retain avid phagocytic and bactericidal activity [1 , 9 , 13 ]. Blood monocytes, which do express CD14 (>96%), lose expression of this receptor following incubation with S-CM in vitro and through the action of stromal-derived TGF-ß, develop into inflammation-anergic macrophages [1 ]. We now demonstrate that blood monocytes, which chemotax to stromal-derived factors, are indeed primarily CD14+, suggesting that in vivo, CD14+ monocytes are recruited to the mucosa and subsequently lose their expression of CD14. The findings presented here suggest the following sequence of events: IL-8 and latent TGF-ß, produced by epithelial cells and mast cells are released into the lamina propria, where they bind to the lamina propria ECM [30 , 31 ]. In this regard, IL-8 has been shown to bind to ECM components, including heparin sulfate and chondroitin sulfate, in other tissues such as the lung [32 , 33 ]. The release of chemokines from the intestinal stroma is likely not unique, as stroma-derived TGF-ß has been implicated in splenic and mammary gland cell functions [34 , 35 ]. In noninflamed intestinal stroma, the TGF-ß, IL-8, and possibly other stromal factors, released in response to local factors such as mast cell chymase [36 ], induce chemotaxis of blood monocytes into the mucosa. Following recruitment, local TGF-ß and other factors induce differentiation of the proinflammatory monocytes into noninflammatory, intestinal macrophages, which express no CD14 and lose their chemotactic capabilities (Fig. 7 ). Taken together, our findings suggest that under noninflamed conditions, newly recruited monocytes acquire a profound inflammation anergy, which limits mucosal inflammation and maintains mucosal homeostasis.


Figure 7
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  		Figure 7. Chemotactic role of ECM (stroma)-derived TGF-ß and IL-8 in the recruitment of blood monocytes into the intestinal mucosa. TGF-ß released by epithelial cells and mast cells and IL-8 released by epithelial cells and mononuclear cells in the lamina propria bind to ECM and serve as chemotactic ligands for proinflammatory CD14+ blood monocytes, which express receptors for TGF-ß, IL-8 (CXCR1,2), C5a, and fMLP. Once recruited, the monocytes lose their ability to chemotax and differentiate into noninflammatory anergic intestinal macrophages.

 
The mechanisms(s) by which chemotaxis-competent monocytes recruited to the mucosa lose their ability to chemotax once they become resident macrophages in the mucosa are unclear. The inability of intestinal macrophages to perform receptor-mediated chemotaxis suggests defective cell locomotion or chemokine receptor-mediated signal transduction. In this regard, we recently identified defects at multiple steps in the signal transduction cascade leading to nuclear factor-{kappa}B activation in intestinal macrophages (L. E. Smythies, manuscript in preparation). Despite profoundly down-regulated chemotaxis, intestinal macrophages display avid phagocytic activity for fungi, inert material, and bacteria [1 , 9 ], indicating that the signal transduction pathways, which regulate new actin filament networks that drive phagocytosis [37 , 38 ], act independently of the pathways that drive chemotaxis [39 , 40 ]. Studies of the signal transduction pathways that induce intestinal macrophage phagocytosis in the absence of IgG receptors [1 ] and C5aR, as shown here, are currently underway. Understanding the regulation of monocyte recruitment to noninflamed and inflamed intestinal mucosa is an important step in devising novel strategies to modulate mucosal inflammation toward inflammatory intestinal diseases.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (DK-74033, DK-47322, DK-54495, HD-41361, DE-16005), the Crohn’s and Colitis Foundation of America, a University-wide Interdisciplinary Research Center grant to the UAB Vision Science Research Center, and the Research Service of the Veterans Administration.

Received October 4, 2005; revised March 7, 2006; accepted April 6, 2006.

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