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Lymphocytes Produce IL-1ß in Response to Fc Receptor Cross-Linking: Effects on Parenchymal Cell IL-8 Release¹

Clay B. Marsh^2,*, Melissa P. Lowe^*, Brad H. Rovin, Jennifer M. Parker^*, Zhiming Liao^*, Daren L. Knoell and Mark D. Wewers^*

Divisions of ^* Pulmonary and Critical Care Medicine and Nephrology, College of Medicine and College of Pharmacy, Ohio State University, Columbus, OH 43210

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neutrophils mediate tissue injury in response to immune complexes, although the factors that induce their recruitment are incompletely understood. We have reported that lymphocytes may be important regulators of monocyte and macrophage IL-8 release in the presence of immobilized IgG. Since tissue parenchymal cells are important local producers of IL-8 but are not directly stimulated by FcR cross-linking, we hypothesized that lymphocytes may also regulate parenchymal IL-8 release. Supernatants from lymphocytes incubated on immobilized IgG induced primary human fibroblasts and human mesangial cells to produce IL-8 (17 ± 3.5 and 44 ± 8 ng/ml, respectively). Fibroblast and mesangial cell IL-8 mRNA levels were similarly increased by the conditioned lymphocyte supernatant. Immobilized anti-human FcRIII, but not FcRI or FcRII Abs, could stimulate this IL-8-inducing activity in lymphocytes, suggesting that FcRIII-bearing lymphocytes were responsible. Supernatants from lymphocytes incubated on immobilized IgG contained 2.2 ± 0.8 ng/ml of IL-1ß, while enriched monocyte preparations from the same donors incubated on immobilized IgG released only 0.1 ± 0.04 ng/ml of IL-1ß (p = 0.05). Consistent with the identification of IL-1ß as the lymphocyte factor, fibroblast or mesangial cell IL-8 release induced by the IgG-stimulated lymphocyte supernatants was inhibited by 1) the combination of IL-1R antagonist and soluble type II IL-1R, 2) an IL-1-converting enzyme inhibitor, or 3) anti-IL-1ß but not preimmune Abs. These data suggest that targeted deposits of IgG can stimulate FcRIII-bearing lymphocytes to produce IL-1ß, which induces parenchymal cell IL-8 release.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immune complexes cause cellular activation by cross-linking surface FcR.³ We have recently demonstrated that lymphocyte FcRIII cross-linking induces the release of a soluble factor capable of stimulating monocyte and macrophage IL-8 expression (1). The relevance of this observation is underscored by the finding that in immune complex-associated diseases such as rheumatoid arthritis, idiopathic pulmonary fibrosis, or glomerulonephritis, tissue injury is associated with localized neutrophil influx (2, 3, 4, 5, 6, 7). This neutrophil influx is dependent on IL-8 production, as biologic fluids from diseased compartments contain large concentrations of the chemokine IL-8 (2, 3, 4, 5, 6, 7), and neutralizing Abs to IL-8 attenuate both the neutrophil influx and tissue injury in response to immune complexes (8, 9).

While the identity of the IgG-stimulated lymphocyte factor is unknown, the majority of lymphocytes that bear FcRIII are NK lymphocytes (10, 11, 12, 13). These NK lymphocytes, often referred to as large granular lymphocytes because of their morphology, are cytokine-producing cells. NK cells have been reported to make IL-8 (14) and IL-1ß (15). We have previously demonstrated that FcRIII-bearing lymphocytes release small concentrations of IL-8 in response to FcR cross-linking; they also release soluble products that induce monocytes to release IL-8 (1, 16) and MCP-1 (17).

In addition to monocytes, tissue parenchymal cells are an important source of tissue IL-8 production in immune complex diseases (18, 19, 20, 21). To date, it is not known how parenchymal cells are induced to release IL-8 in a milieu containing deposits of IgG. Although direct stimulation of chemokine products by FcR stimulation has been documented using rodent mesangial cells (22), those results have not been reproduced in human cells. Additionally, other types of parenchymal cells, such as fibroblasts, which produce IL-8, do not express surface FcR (23). We thus postulate that lymphokines released by cross-linking FcR in lymphocytes could induce parenchymal cell IL-8 release.

To test this hypothesis, primary cultures of human gingival fibroblasts and human renal mesangial cells were incubated with supernatants from lymphocytes that had been cultured on immobilized pooled human IgG. IL-8 mRNA expression and protein production from these parenchymal cells were measured. Additionally, we sought to identify the factor responsible for the IL-8-inducing activity of the cultured lymphocyte supernatants.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

Recombinant human IL-1ß was a gift from the Biologic Response Modifiers Branch, National Cancer Institutes (Bethesda, MD); RPMI 1640 medium was obtained from BioWhittaker (Walkersville, MD); 5% FCS was obtained from HyClone (Logan, UT); anti-human FcR Abs, anti-FcRI (clone 197, IgG2a isotype), anti-FcRII (Fab clone IV.3), and anti-FcRIII (F(ab')₂ clone 3G8), were a gift from Medarex (West Lebanon, NJ); polymyxin B was obtained from Rohrer Pharmaceuticals (New York, NY); anti-IL-1ß mAb was a gift from Dr. Ann Berger, Upjohn Laboratories (Kalamazoo, MI); Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD-CMK) was obtained from Calbiochem (Cambridge, MA); recombinant human IL-1Ra used to make polyclonal Abs against IL-1Ra was a gift from Dr. Daniel Tracey, Upjohn Laboratories; goat anti-human IL-1Ra, mouse anti-human IL-8, and rabbit anti-human IL-8 Abs were obtained from R&D Systems (Minneapolis, MN); magnetic beads were obtained from Dynal (Lake Success, NY); a partial cDNA probe for IL-8 was a gift from Genentech (South San Francisco, CA).

Cell purification

Mononuclear cells from heparinized venous blood of normal human volunteers were purified and enriched for lymphocytes as previously detailed (1, 16). Lymphocytes (5 x 10⁶ lymphocytes/ml) were resuspended in RPMI 1640 medium with 5% FCS and polymyxin B (10 µg/ml). As previously shown, this concentration of polymyxin B is sufficient to completely inhibit LPS (10 ng/ml)-induced IL-8 in PBMC and does not affect cell viability or IL-8 production (24). Using FACS analysis, purified lymphocytes were 1.7 ± 0.6% CD14⁺/CD16^-, 0.2 ± 0.8% CD14⁺/CD16⁺, 14.5 ± 0.9% CD14^-/CD16⁺, and 83 ± 12% CD14^-/CD16^-. In contrast, monocyte preparations were 62 ± 1.5% CD14⁺/CD16^-, 5.6 ± 2.6% CD14⁺/CD16⁺, 7.8 ± 0.8% CD14^-/CD16⁺, and 26 ± 4% CD14^-/CD16^-. Thus, at the cell concentrations used in these experiments, lymphocyte preparations had a 10-fold greater concentration of FcRIII-bearing lymphocytes than did monocyte preparations (7.2 x 10⁵ CD14^-/CD16⁺ cells/ml vs 7.8 x 10⁴ CD14^-/CD16⁺ cells/ml, respectively).

In separate experiments, magnetic beads coated with anti-FcRIII Abs (clone 3G8) were used to select FcRIII-bearing lymphocytes from the nylon wool preparations. A bead:cell ratio of 4:1 was used in these experiments (0.02 ml of the FcRIII-coated beads (12 x 10⁸/ml) were added to 0.5 ml of lymphocytes (10 x 10⁶/ml) and rotated for 45 min at 4°C on an orbital rotator). Selected cells were separated using a magnet, resuspended in 0.5 ml of medium, and incubated on immobilized IgG for 18 h as previously described. The unselected cells were also resuspended in 0.5 ml of media and incubated on immobilized IgG for 18 h. Cell-free supernatants were then recovered and assayed for IL-1ß by ELISA. Cell staining using Abs staining for FcRIII revealed that the FcRIII-coated beads isolated 99% of the FcRIII-bearing lymphocytes (14.5 ± 0.9 to 0.045 ± 0.05%).

Parenchymal cell preparation

Primary human gingival fibroblasts (0.24 x 10⁶/well) were purified from extracted wisdom teeth (passages 3–5) and suspended in DMEM, 10% FCS, gentamicin, and fungizone. Freshly isolated human mesangial cells were obtained from four normal human donor kidneys that were not used for transplant because of prolonged time ex vivo. These mesangial cell cultures were free of contaminating glomerular capillary endothelial cells or glomerular epithelial cells. Mesangial cells were suspended in RPMI 1640 medium, 10% FCS, and gentamicin and plated overnight until confluent on tissue culture plates.

Culture conditions

Purified lymphocytes were incubated for 18 h on 1) plastic alone, 2) immobilized pooled human IgG (25 µg plating concentration/well), 3) immobilized whole molecule anti-human FcRI (clone 197), 4) immobilized Fab anti-FcRII (clone IV.3), or 5) immobilized F(ab')₂ anti-FcRIII (clone 3G.8) Abs (25-µg plating concentration/well). Of note, we have previously demonstrated that these immobilized Abs are biologically active (1), suggesting that differential induction of the lymphocyte preparations is a reflection of specific FcR engagement. These Abs were immobilized on tissue culture dishes coated for high efficiency protein binding (Immulon IV, Dynatech, Chantilly, VA) as previously described (16). Cell-free supernatants from these cells (1/5 dilution) were incubated with human gingival fibroblasts or mesangial cells. In some experiments, the combination of IL-1Ra (10 ng/ml) and soluble type II IL-1R (10 ng/ml) was added to the fibroblasts or mesangial cell cultures.

In other experiments, purified lymphocytes were incubated on immobilized human pooled IgG (25-µg plating concentration/well) for 18 h in the presence or absence of YVAD (0.01–100 µM), an inhibitor of IL-1-converting enzyme (ICE). The cell-free supernatants from these cells were then incubated with the parenchymal cells.

Lastly, IgG-stimulated lymphocyte supernatants were incubated with protein A beads alone or with protein A beads that were bound to either rabbit polyclonal anti-IL-1ß or preimmune rabbit IgG overnight at 4°C, and then the protein A or protein A/IgG complexes were removed by centrifugation. Lymphocyte supernatants incubated on plastic tissue culture plates alone were also incubated with protein A beads overnight at 4°C, and the beads were removed by centrifugation. These treated lymphocyte supernatants were then incubated with human gingival fibroblasts or human mesangial cells (1/5 dilution) for an additional 18 h at 37°C in 5% CO₂.

All reagents used in these experiments contained <10 pg/ml of contaminating LPS, as assessed by chromogenic Limulus amebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA), and media for all cell preparations contained polymyxin B.

IL-8 and IL-1ß ELISAs

As previously described, specific ELISAs for IL-8 (16), IL-1Ra (24), and IL-1ß (24) were used to assay cell-free supernatants. The IL-8 assay is sensitive to 300 pg/ml, and the IL-1ß assay is sensitive to 30 pg/ml. Importantly, the combination of IL-1Ra and soluble type II IL-1R did not augment or interfere with detection of rIL-8 in the IL-8 ELISA.

Results given for IL-8 cellular production of fibroblasts and mesangial cells stimulated with IgG- or plastic-stimulated lymphocyte supernatants were reported as the total amount of IL-8 detected minus the IL-8 contained in a 1/5 dilution of the lymphocyte supernatants (IgG-stimulated lymphocytes, 6 ± 4 ng/ml of IL-8; adherent lymphocytes, <0.3 ng/ml of IL-8).

RNA extraction and purification

Total cellular RNA was purified from human gingival fibroblasts or human mesangial cells (grown to confluence) that had been incubated for 18 h with conditioned lymphocyte supernatants at 37°C in 5% CO₂ as previously described (16, 25). IL-8 mRNA was identified using a 478-bp IL-8 cDNA that was labeled using ³²P by random priming using Northern analysis and autoradiography.

FACS staining

FACS staining was performed to confirm that lymphocytes, rather than contaminating monocytes, produced IL-1ß and surface CD14. Briefly, lymphocytes purified on nylon wool (1.2% CD14⁺, 20% FcRIII⁺ cells by FACS) were incubated on immobilized IgG (25-µg plating concentration/well) for 18 h at 37°C in 5% CO₂. The cells were recovered, washed, and stained initially with CD14 Abs, a monocyte-specific marker, then fixed with 2% paraformaldehyde, permeabilized using saponin, and stained for intracellular IL-1ß using a monoclonal anti-IL-1ß Ab. Parallel samples of the cells were stained with isotype control Abs for CD14 and IL-1ß to account for nonspecific staining of the cells. The cells were sorted on an Elite I flow cytometer (Coulter, Hialeah, FL) and interpreted using Image-1/Metamorph imaging software (Universal Imaging Corporation, West Chester, PA). All FACS analysis was performed at the Ohio State University Analytical Cytometry Laboratory (Columbus, OH).

To ensure that permeabilization with the saponin did not cause surface CD14 receptors to be shed, control experiments with freshly isolated, enriched monocyte preparations were performed and demonstrated that staining of CD14 receptors was not affected by the saponin permeabilization.

Statistical analysis

To compare more than two conditions, ANOVA with Fisher’s post hoc testing was used (Minitab, State College Park, PA). Dose-response curves were analyzed by linear regression using Fig P (Biosoft, Ferguson, MO) software. Results are expressed as the mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Supernatants from IgG-stimulated lymphocytes induce fibroblasts and mesangial cells to release IL-8

To determine whether conditioned lymphocyte supernatants can induce tissue parenchymal cells to release IL-8, supernatants from nylon wool-purified lymphocytes (98% CD14^- by FACS) incubated on immobilized IgG were incubated with parenchymal cells. These supernatants stimulated the production of 17 ± 3.5 ng/ml of IL-8 from primary gingival fibroblasts, while supernatants from lymphocytes incubated on plastic alone caused no detectable fibroblast IL-8 release (p < 0.0001; n = 8). Similarly, primary human renal mesangial cells stimulated with supernatants from IgG-treated lymphocytes (1/5 dilution) released 44 ± 8 ng/ml of IL-8, while those treated with supernatants from lymphocytes incubated on plastic alone released only 7.1 ± 1.4 ng/ml (p = 0.01; n = 10). Importantly, fibroblasts incubated for 18 h on immobilized IgG did not release detectable amounts of IL-8, and mesangial cells incubated on immobilized IgG did not release more IL-8 than did mesangial cells incubated on plastic alone (1.8 ± 0.7 vs 1.1 ± 0.5 ng/ml; n = 3), suggesting that parenchymal cell IL-8 induction was not due to immune complexes that may have been present in the lymphocyte supernatants.

IgG-stimulated lymphocytes release IL-1ß

To identify the chemokine-inducing activity found in immobilized IgG-stimulated lymphocyte supernatants, we measured several candidate cytokines in 18-h cell-free lymphocyte supernatants, including IL-1ß. Immobilized IgG induced lymphocytes to release 2.2 ± 0.8 ng/ml of IL-1ß. In contrast to the enriched lymphocyte preparations, enriched monocyte preparations from the same individuals released only 0.1 ± 0.04 ng/ml of IL-1ß (p = 0.05 for monocytes compared with lymphocyte preparations; n = 4; Fig. 1A).

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Comparison of monocyte and lymphocyte cytokine release by immobilized IgG. A, IL-1ß and IL-1Ra release from nylon wool-purified human lymphocytes (5 x 106 ml) containing 9 x 104 monocytes/ml or from enriched monocyte preparations (1 x 106 monocytes/ml) from the same individuals was determined by ELISA after incubation on immobilized IgG. The data represent the mean ± SEM for four separate individuals. B, FcRIII+ and FcRIII- lymphocytes from the same individuals were separated using FcRIII-coated magnetic beads. The FcRIII+ and FcRIII- cells were plated on immobilized human IgG for 18 h, and IL-1ß was then measured on cell-free supernatants. The data shown represent paired experiments for five individuals.

FcRIII-bearing lymphocytes appeared to be the primary source of IL-1ß secretion, as they released 2.6 ± 1 ng/ml of IL-1ß vs 0.6 ± 0.6 ng/ml for FcRIII-negative lymphocytes (p = 0.028, by paired t test; Fig. 1B). In composite, these data suggest that FcRIII-bearing lymphocytes were induced to release IL-1ß in response to FcR cross-linking.

Of interest, we found that IgG-stimulated lymphocyte supernatants at 1/5 dilution (0.5 ng/ml of IL-1ß) induced fibroblasts to release 17 ± 3.5 ng/ml and MC to release 44 ± 8 ng/ml of IL-8. In contrast, recombinant human IL-1ß (0.1 ng/ml) stimulated fibroblasts and mesangial cells to release 38 ± 6 and 62 ± 8 ng/ml of IL-8, respectively. These data are consistent with the complicated nature of biologic supernatants from the IgG-stimulated lymphocytes, which undoubtedly contain additional modifying factors.

Since IL-1ß is produced as a cell-associated protein before being released, we double labeled cells in lymphocyte preparations with an anti-IL-1ß Ab to detect intracellular IL-1ß and a surface anti-CD14 Ab to detect monocytes, to identify the IL-1ß-producing cell type. This analysis demonstrated that of cells in the lymphocyte preparation, immobilized IgG induced 69% of the CD14-negative cells (nonmonocytes) to produce intracellular IL-1ß (Fig. 2C). In contrast, <1% of the total lymphocyte population of CD14-positive cells (monocytes) stained positive for IL-1ß (Fig. 2C). Consistent with a specific induction by immobilized IgG, <2% of the total lymphocyte population stained for intracellular IL-1ß when incubated on plastic tissue culture dishes alone (Fig. 2B). To confirm that these IgG-stimulated lymphocytes were producing IL-1ß, as opposed to secondarily accumulating released IL-1ß, we repeated the analysis using a pro-IL-1ß Ab specific for the precursor form of IL-1ß. Consistent with newly formed intracellular IL-1ß, the polyclonal rabbit Ab to pro-IL-1ß also stained CD14-negative cells after incubation on immobilized IgG (data not shown). Additionally, when an anti-CD64 Ab was used to identify monocytes, IL-1ß staining was still localized to the lymphocyte populations (data not shown).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Lymphocytes are the source of the IL-1ß production. To exclude contaminating monocytes (<2% of the population) as the source of the observed release of IL-1ß from nylon wool-purified lymphocyte cultures, cells were analyzed for intracellular IL-1ß and surface CD14 staining by FACS. Purified lymphocytes that had been dually stained with FITC-labeled IL-1ß (ordinate) and phycoerythrin (PE)-labeled CD14 (abscissa) were analyzed. Two percent of the cell stimulated on plastic for 18 h showed IL-1ß staining (B). In contrast, when lymphocytes were incubated on immobilized IgG for 18 h, 70% of the CD14-negative cells and <1% of the CD14-positive cells stained positive for IL-1ß (C). These data are representative of four experiments using different subjects.

Lymphocyte FcRIII induces the secretion of parenchymal cell IL-8 release

To further determine whether lymphocyte FcR were responsible for the induction of parenchymal cell IL-8-stimulating activity, immobilized monoclonal anti-human FcR Abs were used to stimulate lymphocytes. Consistent with our previous observations, supernatants from lymphocytes stimulated with immobilized anti-human FcRIII Abs induced 96% more IL-8 release from fibroblasts than did supernatants from lymphocytes exposed to anti-FcRI or anti-FcRII Abs (p = 0.001; Table I).

View this table: [in this window] [in a new window]   Table I. Ability of specific FcR receptors to induce IL-8 releasing factor from lymphocytes1

To further examine whether IL-1ß was responsible for the lymphocyte activity, a combination of soluble type II IL-1R and IL-1Ra (at 10 ng/ml each) was added to conditioned lymphocyte supernatants to prevent the interaction of IL-1 with the type I signaling IL-1R. These IL-1ß inhibitors effectively blocked IL-8 release from the parenchymal cells, suggesting that IL-1ß was the source of the chemokine-inducing activity (Fig. 3). Fibroblast IL-8 release declined from a baseline of 17 ± 3.5 to 2.2 ± 1.5 ng/ml (p = 0.0001, n = 8), and mesangial cell IL-8 release declined from a baseline of 44 ± 8 to 9.2 ± 3.5 ng/ml (p = 0.001; n = 10; Fig. 3). Moreover, these IL-1 inhibitors also suppressed FcRIII-stimulated lymphocyte supernatant induction of fibroblast IL-8 release (Table I).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Inhibitors of IL-1ß decrease IL-8 induction in parenchymal cells in response to rIL-1ß or IgG-stimulated lymphocyte supernatants. Human gingival fibroblasts (A) or human mesangial cells (B) were incubated with 1) recombinant human IL-1ß (0.1 ng/ml for fibroblasts and 1.1 ng/ml for mesangial cells), 2) recombinant human TNF- (100 ng/ml), 3) immobilized IgG-stimulated lymphocyte supernatants, or 4) supernatants from lymphocytes stimulated by incubation on plastic culture plates alone. These incubations were performed in the presence or the absence of the combination of IL-1Ra and soluble type II IL-1R (both at 10 ng/ml, IL-1 inhibitors). Cell-free supernatants were assayed for IL-8 by ELISA. These data represent the mean ± SEM for fibroblasts (n = 8) and mesangial cells (n = 10).

Since IL-1ß is produced as an inactive precursor molecule and requires processing by ICE for activity, we next attempted to block ICE activity by incubating the immobilized IgG-stimulated lymphocytes with the synthetic tetrapeptide ICE inhibitor, YVAD-CMK. Supernatants from lymphocytes treated with this ICE inhibitor showed a dose-dependent reduction in the ability to stimulated parenchymal cell IL-8 expression (n = 3 for fibroblasts and n = 2 for mesangial cells; Fig. 4).

View larger version (14K): [in this window] [in a new window]   FIGURE 4. ICE inhibitors block the release of IL-8-inducing activity by purified lymphocytes stimulated on immobilized human IgG. Nylon wool-purified lymphocytes (5 x 106/ml) were incubated on immobilized human IgG (0.1 mg/ml) in the presence or the absence of YVAD-CMK (0.01–100 µM) and inhibitor of ICE or on plastic plates alone for 18 h in 5% CO2 at 37°C. The cell-free supernatants were recovered, and IL-8 was assayed by ELISA. YVAD-CMK inhibited fibroblast IL-8 release at a dose of 100 µM (p < 0.03) and mesangial cell IL-8 release at doses of 10 and 100 µM (p < 0.03). This represents the mean ± SEM for fibroblasts (n = 3) and mesangial cells (n = 2).

We next determined whether blocking the effects of IL-1ß suppressed fibroblast and mesangial cell IL-8 mRNA induction by supernatants from immobilized IgG-stimulated lymphocytes. As shown in Figure 5, A and B, the combination of IL-1Ra and soluble type II IL-1R decreased 18-h mesangial cell IL-8 steady state mRNA levels that had been induced by either rIL-1ß or supernatants from immobilized IgG-stimulated lymphocytes. Figure 5C demonstrates a similar effect on primary human gingival fibroblasts. To account for loading differences, laser densitometry was performed, comparing IL-8 mRNA to GAPDH controls (Fig. 5D). These studies confirmed that the IL-1 inhibitors were able to suppress IL-8 steady state mRNA induced by either rIL-1ß or the IgG-stimulated lymphocyte supernatants. In contrast, these inhibitors did not suppress rTNF--induced steady state IL-8 mRNA. (Note, the concentration of human rIL-1ß used to stimulate fibroblasts was 1/10th that used to stimulate mesangial cells, which may account for differences in the levels of IL-8 mRNA in these blots.) In composite, these data further document that lymphocytes incubated on immobilized IgG release IL-1ß, which can induce parenchymal cell IL-8 release.

View larger version (39K): [in this window] [in a new window]   FIGURE 5. IL-1ß inhibitors suppress gingival fibroblast and human mesangial cell steady state IL-8 mRNA induced by IL-1ß or supernatants from immobilized IgG-stimulated lymphocytes. Human mesangial cells (A and B) or human gingival fibroblasts (C) were cultured as described in Figure 2. After 18-h incubations, total RNA was recovered, and IL-8 mRNA was identified using a full-length IL-8 probe. The IL-1ß inhibitors (INH), soluble type II IL-1R and IL-1Ra (10 ng/ml each), suppressed IL-8 mRNA from mesangial cells or fibroblasts treated with either recombinant human IL-1ß (0.1 ng/ml) or IgG-stimulated lymphocyte supernatants (LYG), but did not suppress mesangial cell or fibroblast IL-8 mRNA stimulated by recombinant human TNF-. D, Densitometric ratios of IL-8 steady state mRNA to GAPDH control lanes to account for differences in loading the lanes. The results shown are representative of two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To characterize the lymphocyte factor that induces IL-8 production, we found that lymphocytes incubated on immobilized IgG released sufficient amounts of IL-1ß to induce IL-8 in both fibroblasts and mesangial cells. Consistent with IL-1ß generation by the IgG-stimulated lymphocytes, supernatants from these IgG-stimulated lymphocytes cultured in the presence of the ICE inhibitor YVAD-CMK lost their ability to induce parenchymal cell IL-8 release. Using a combination of the IL-1ß inhibitors, IL-1Ra and soluble type II IL-1R, which are synergistic in blocking IL-1-induced target cell activation (26), we found that the combination of these two IL-1ß inhibitors inhibited fibroblast IL-8 release by 85% and mesangial cell IL-8 release by 73% in response to IgG-stimulated lymphocyte supernatants. Additionally, neutralizing anti-IL-1ß, but not preimmune Abs, also inhibited IgG-stimulated lymphocyte supernatant induction of fibroblast or mesangial cell IL-8 release. In aggregate, these data argue that the IL-8 activity in the lymphocyte supernatants is largely IL-1ß.

We next wanted to determine whether lymphocytes or the small numbers (<2%) of contaminating monocytes were the cells producing IL-1ß in response to FcR cross-linking. We found that despite having only 1/10th as many monocytes, lymphocyte preparations released 100-fold more IL-1ß than autologous monocyte preparations. In contrast, the monocytes were still able to respond to FcR cross-linking, as autologous monocyte preparations released 10-fold more IL-1Ra than did lymphocyte preparations. Moreover, by flow cytometry, we found that CD14-negative cells appeared to be the cellular source of IL-1ß induced by FcR cross-linking. As stimulation of lymphocyte FcRIII, but not FcRI or FcRII, caused the release of IL-8-inducing activity, we surmised that FcRIII-bearing lymphocytes are the source of IL-1ß. This concept is supported by finding that FcRIII⁺ lymphocytes release more IL-1ß than FcRIII^- cells, which supports previous observations from our laboratory (1, 27). However, we cannot definitively rule out some interaction between the small number of monocytes and lymphocytes in the release of IgG-stimulated IL-1ß.

It is particularly noteworthy that FcRIII-bearing lymphocytes are expanded in immune complex-related diseases (28, 29, 30). For example, FcRIII-bearing lymphocytes represent 20 to 80% of the mononuclear cells in the rheumatoid synovium, as opposed to approximately 2% of the normal synovial mononuclear cell population (28). Although the role of this cadre of FcRIII-bearing lymphocytes is not known, we speculate that they may play a regulatory role in modulating leukocyte influx through inducing monocyte and parenchymal cell chemokine production. While other investigators have found that large granular lymphocytes can release IL-1ß (15, 31, 32), we believe that this is the first demonstration that FcR cross-linking can stimulate this release. This observation has relevance to other pathophysiologic conditions, including the association of large granular lymphocytosis and neutropenia (33, 34, 35, 36). We hypothesize that these patients may have unregulated secretion of IL-1ß, which may augment IL-8 secretion by tissue cells, causing neutrophil egress from the intravascular to extravascular space.

The present observations extend our previous work (1, 16, 27) by showing both that lymphocyte FcRIII cross-linking induces parenchymal cells to release biologically active IL-1ß, but also that lymphocyte FcR cross-linking can induce resident parenchymal cells to release chemokines in a paracrine manner. Although parenchymal cells have been identified as an important source of tissue-generated IL-8, the mechanism of this production is unknown. In the setting of IgG deposition in the tissue, this study suggests that circulating lymphocytes may be able to recognize this IgG and induce neutrophil recruitment via parenchymal cell IL-8 production. This parenchymal cell production would induce a natural gradient for the neutrophils to egress into the involved tissue. Because such a gradient is needed for neutrophil migration into the tissue compartment in response to IL-8 (37), this pathway may be a critical physiologic mechanism by which lymphocytes may be able to induce and amplify tissue injury in immune complex diseases.

	Acknowledgments

 
We thank the Analytical Flow Cytometry Laboratory at Ohio State University and Joe Trask for assistance with this project, and Dr. Alissa Winnard for her assistance and helpful suggestions.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HL40871, HL140150, M01RR00034–36S2, and DK46055; the American Lung Association of Ohio; a Parker B. Francis Families Fellowship in Pulmonary Research (to C.B.M.); and a Clinical Scientist Award from the National Kidney Foundation (to B.H.R.).

² Address correspondence and reprint requests to Dr. Clay B. Marsh, Division of Pulmonary and Critical Care Medicine, Ohio State University, Columbus, OH 43210. E-mail address:

³ Abbreviations used in this paper: FcR, Fc receptor; YVAD, tyrosine-valine-alanine-aspartate-cholormethylketone; CMK, chloromethylketone; IL-1Ra, IL-1 receptor antagonist; CD14, monocyte-specific receptor; CD16, FcRIII; ICE, IL-1-converting enzyme; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Received for publication July 29, 1997. Accepted for publication December 12, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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