Introduction

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The acute inflammatory reaction is a complex response that the host mounts against external intrusions and is essential for the survival of the organism. However, the question as to why a beneficial process such as inflammation becomes disregulated to the point of causing disease, i.e., when inflammation becomes chronic, is still unanswered. Chronicity is defined by the continual accumulation and activation of inflammatory cells at the tissue site, and the self-perpetuating nature of this event is supported by the fact that in many instances, no etiologic or antigenic element can be found to account for the continual presence of inflammation. Thus, modulation of the inflammatory response and, specifically, how inflammation evolves into a chronic and self-perpetuating process remain largely to be clarified. The host response to injury involves a complex set of cell-to-cell interactions and a variety of signals at the cellular level. Among these signals, cell-derived cytokines play a fundamental role. Traditionally, immune effector cells such as lymphocytes and macrophages have been considered the sources of these molecules. A great deal of evidence is now available showing that resident structural cells, such as fibroblasts, epithelial and endothelial cells as well as smooth muscle cells, are also capable of releasing cytokines and are therefore considered effector cells (1). Particularly, recent studies demonstrate that pulmonary fibroblasts, far from being merely bystander cells, are able to affect inflammatory and immune responses through the secretion of prostaglandins (PGs), cytokines, and growth factors (8).

We have already shown that human lung fibroblasts inhibit the production of tumor necrosis factor (TNF)- by lipopolysaccharide (LPS)-activated peripheral blood monocytes through the release of soluble factors such as PGE₂ (12, 13), and have hypothesized that during the reparative process after inflammation, activated monocytes produce an increased amount of proinflammatory cytokines, including TNF-. These cytokines can directly stimulate fibroblast functions and also induce these cells to produce increased amounts of PGE₂ that can in turn exert a negative feedback on mononuclear phagocyte cytokine production as well as an autocrine inhibition of fibroblast proliferation and metabolic functions, leading to self-limitation of the reparative and inflammatory process (14, 15). Chronic inflammation may indeed represent the result of a failure in this regulatory mechanism: unrestrained activation or defective suppression in one or more of the cells involved could in fact result in an imbalance leading to disease. In this study, we have tried to determine whether fibroblasts may contribute to the transformation of the acute inflammatory response into a chronic process. To address this question, we looked at some functional features of fibrotic human lung fibroblasts as compared with normal cells. As we have already shown (12), the interaction between normal fibroblasts and monocytes appears to be addressed to a mutual control of their activities; interestingly, fibrotic cells do not show this capability. Our data indicate that fibroblasts coming from fibrotic tissue loose their ability to interact with monocytes because they produce less PGE₂ compared with normal cells, and as a consequence of that, they show a reduced inhibitory action in the production of TNF- by monocytes. In addition, owing to the significant reduction of the expression of TNF receptors (TNFRs), fibrotic cells exhibit a modified response to TNF exposure. These results support the hypothesis that the emergence, in the context of inflammation, of cells with distinct phenotypic features would introduce into the system different patterns of cell-to-cell interaction that might be crucial for the evolution of the inflammatory process.

	Materials and Methods

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Fibroblast Cultures

Primary lines of normal human adult lung fibroblasts (NF) were established by using an outgrowth from explant according to the method described by Jordana and coworkers (16). Fibroblast lines were derived from histologically normal areas of surgical lung specimens from patients undergoing resective surgery for cancer. Their ages ranged from 52 to 61 yr. Five of six patients were men. Lung specimens were chopped into pieces of less than 1 mm³ and washed once with phosphate-buffered saline (PBS) and twice with RPMI containing 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml fungizone (supplemented RPMI) (GIBCO, Paisley, UK). Eight to ten pieces of washed specimens were then plated in a 100-mm polystyrene dish (Falcon; Becton Dickinson, Lincoln Park, NJ) and overlaid with a coverslip held to the dish with sterile vaseline. Ten milliliters of supplemented RPMI were added and the tissue was incubated at 37°C with 5% CO₂. The medium was changed weekly. When a monolayer of fibroblast-like cells covered the bottom of the dish, usually 5 to 6 wk later, the explant tissue was removed, and the cells were then trypsinized for 10 min, resuspended in 10 ml of supplemented RPMI, and plated in 100-mm tissue culture dishes. Subsequently, cells were split 1:2 at confluence, usually weekly. Aliquots of cells were frozen and stored in liquid nitrogen. Fibrotic lung fibroblast lines (FF) were established from histologically proven fibrotic lung tissue of patients with interstitial lung disease and were kindly provided by Dr. Manel Jordana (Department of Pathology, McMaster University, Hamilton, ON, Canada). The ages of patients with lung fibrosis ranged from 45 to 55 yr, except one patient who was a 12-yr-old subject. In the considered parameters, the cell line derived from this patient did not behave differently compared with the other cell lines and for this reason was included in the study. Five of six patients were men. In all experiments, we used cell lines at a passage earlier than the tenth.

Monocyte Isolation Procedure

Heparinized venous blood, obtained from healthy donors, was diluted 1:4 with PBS, and 40 ml were then placed on 10 ml of Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) for centrifugation at 1,600 rpm for 20 min at room temperature. Mononuclear cells were collected at the interface, washed twice, and resuspended in PBS supplemented with 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid. Isolation of human monocytes from mononuclear cells was performed by depletion of nonmonocytes using a magnetic cell sorting system (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. T cells, natural killer cells, B cells, dendritic cells, and basophils were indirectly magnetically labeled using a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-immunoglobulin (Ig) antibodies and magnetic microbeads coupled to an antihapten monoclonal antibody. The magnetically labeled cells were depleted by retaining them on a column in the magnetic field of the MACS system. Unlabeled cells, representing the enriched monocyte fraction, passed through the column and were collected as effluent.

Monocyte Cultures

Peripheral blood monocytes (PBMs) were incubated in 24-well tissue culture plates (Falcon; Becton Dickinson) at a concentration of 2.5 × 10⁵ cells in 1 ml either of fibroblast-conditioned medium (FCM) or supplemented RPMI. LPS from Escherichia coli 0.26:B6 (Sigma) was immediately added (10 µg/ml), and the plates were incubated in a humidified atmosphere of 5% CO₂ at 37°C. After 18 h, supernatants were harvested, centrifuged, stored in 0.5-ml aliquots at 70°C, and then assayed for TNF-.

Fibroblast-Conditioned Medium

Normal (NFCM) and fibrotic (FFCM) fibroblast-conditioned medium were generated from cultures of 2 × 10⁶ fibroblasts incubated for 24 h in 10 ml of supplemented RPMI. Supernatants were centrifuged and stored in aliquots at 70°C until use.

PGE₂ and TNF- Assay

Concentrations of PGE₂ in FCM were determined by a commercially available enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI). The assay is sensitive to 3.9 pg/ml of PGE₂. Concentrations of TNF- in PBM supernatants were also determined by a commercially available enzyme immunoassay (BioSource International, Camarillo, CA) sensitive to 10 pg/ml of TNF-. Both assays were performed according to the manufacturer's instructions, and all samples were determined in duplicate.

Matrix Metalloproteinase 1 Assay

To measure the secretion of matrix metalloproteinase (MMP)-1 into the culture media the cells were maintained in serum-free medium for 18 h. Thereafter, TNF- (20 ng/ml) was added and incubation continued for 48 h. The conditioned media were collected and the amount of MMP-1 was determined using a commercially available enzyme immunoassay (MMP-1 human enzyme-linked immunosorbent assay system, Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer's instructions. The assay is sensitive to 1.7 ng/ml. All samples were determined in duplicate.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) proliferation assay is based on the conversion by mitochondrial dehydrogenases of the substrate containing a tetrazolium ring into blue formazan, detectable spectrophotometrically. The level of blue formazan is then used as an indirect index of cell density. Briefly, after treatment with TNF- (20 ng/ml/48 h), cells were exposed to MTT (5 mg/ml, Sigma) for 150 min at 37°C. Medium was then removed and cells were solubilized with acidified isopropanol and 2% sodium dodecyl sulfate (Sigma). After complete solubilization, the formation of blue formazan was evaluated spectrophotometrically with a reference wavelength of 650 nm.

Cytofluorimetric Assessment of Receptors for TNF

Fibroblasts grown to subconfluence were harvested from the dishes and fixed with 2% paraformaldehyde at 4°C for 30 min. Cells were then repeatedly washed with phosphate buffer and incubated for 45 min at 4°C with goat antihuman TNFR1 or TNFR2 (both at a concentration of 2 µg/ml; Santa Cruz Biotechnologies, Santa Cruz, CA). Both antibodies recognize a specific sequence at the carboxy-terminal portion of each receptor. This step was followed by incubation with fluorescein isothiocyanate (FITC)- conjugated antigoat IgG (Santa Cruz Biotechnologies) for 45 min at 4°C. Controls included omission of the primary antibody and substitution with nonimmune serum. Samples were analyzed using a Coulter Epics Elite ESP flow cytometer (Coulter Corp., Miami, FL). At least 10,000 forward and side scatter gated events were collected per specimen. Cells were excited at 488 nm and the fluorescence was monitored at 525 nm. FITC fluorescence was collected using logarithmic amplification.

Western Blot Analysis of the Expression of Cyclooxygenase 1 and 2

Fibroblasts were treated with TNF- (20 ng/ml) for 4 h, then harvested at 4°C in a lysis buffer containing phenylmethylsulfonyl fluoride, aprotinin, pepstatin A, and leupeptin. After centrifugation at 15,000 × g at 4°C, the supernatant was processed for protein concentration according to the method of Bradford (17). Samples were diluted in sample buffer and boiled for 5 min. Electrophoresis was performed in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (30 mA/h) using 20 µg of total protein per lane. After separation, proteins were transferred onto a nitrocellulose membrane for 45 min at room temperature using a transblot semidry transfer cell. After blocking, the membrane was incubated with goat anticyclooxygenase (COX)-1 and anti-COX-2 (2 µg/ml; Santa Cruz Biotechnologies) for 2 h at room temperature, then repeatedly washed and exposed to horseradish peroxidase-conjugated antigoat IgG (1:2000; Santa Cruz Biotechnologies) for 1 h at room temperature. Proteins were visualized using an enhancing chemiluminescence detection system (Amersham Italia, Milan, Italy).

CD49b (₂₁ Integrin) Expression

CD49b expression was studied in six cell lines of NF and FF. Fibroblasts (4 × 10⁵ cells) were incubated with TNF- (20 ng/ml) for 7 d. Cells were then repeatedly washed with phosphate buffer and incubated for 45 min at 4°C with a mouse antihuman CD49b (PharMingen, San Diego, CA) conjugated with FITC. Samples were analyzed using a Coulter Epics Elite ESP flow cytometer (Coulter Corp.). At least 10,000 forward and side scatter gated events were collected per specimen. Cells were excited at 488 nm and the fluorescence was monitored at 525 nm. FITC fluorescence was collected using logarithmic amplification.

Statistical Analysis

Results are presented as the mean ± standard error (SE). Statistical comparisons between normal and fibrotic cell lines in terms of PGE₂ production and TNFR expression were performed by a nonpaired Student's t test. Percent change of proliferation, TNF- inhibition, CD49 expression, and MMP-1 production in NF and FF were compared with a Mann-Whitney U test for unpaired data. A P value of less than 0.05 was considered significant.

	Results

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Production of PGE₂ by Normal and Fibrotic Human Lung Fibroblasts

The amount of PGE₂ spontaneously released and detected in NFCM was 7,500 ± 270 pg/ml, whereas 3,300 ± 410 pg/ ml were found in FFCM after 24 h of incubation (P < 0.001) (Figure 1A). Values are represented as individual data points and mean of four different FCMs generated by different cell lines of NF and FF. The different kinetics in the spontaneous production of PGE₂ by NF and FF are shown in Figure 1B.

View larger version (15K): [in this window] [in a new window]   Figure 1.   (A) PGE2 content in FCM produced by normal (NFCM) and fibrotic human lung fibroblasts (FFCM). FCM was generated by 2 × 106 cells incubated for 24 h in 10 ml of supplemented RPMI. Values are represented as individual data points and are the means of four different cell lines of normal and fibrotic human lung fibroblasts. (B) Time course of the spontaneous release of PGE2 from one normal (solid squares) and one fibrotic (solid triangles) cell line of fibroblasts. Data are from one representative experiment performed in triplicate.

Production of TNF- by LPS-Stimulated PBM Cultured with FCM

The effect of FCMs on the production of TNF- by LPS-activated monocytes is shown in Figure 2A. FCMs produced by six different cell lines of NF and FF were tested; each FCM was tested at least three times. The percentage of inhibition induced by NFCM was 61 ± 5.9%, whereas the inhibitory effect exerted by FFCM was 32 ± 4%. The statistical analysis showed a significant difference between the percent inhibition induced by NFCM and FFCM (P < 0.01). The correlation between the amount of PGE₂ produced by NF and FF and the percentage of TNF- inhibition induced (r = 0.9, P < 0.01) are shown in Figure 2B.

View larger version (14K): [in this window] [in a new window]   Figure 2.   (A) Effect of FCM derived by normal (NFCM) and fibrotic human lung fibroblasts (FFCM) on the production of TNF- by LPS-activated monocytes. PBM were incubated at a concentration of 2.5 × 105 cells in 1 ml of either FCM or supplemented RPMI with 10 µg/ml of LPS. Values are expressed as percentage of TNF- inhibition and are represented as individual data points and the means of six different cell lines of normal and fibrotic cells. (B) Correlation between the amount of PGE2 produced by normal and fibrotic cell lines of fibroblasts and the percentage of TNF- inhibition induced.

TNFR Expression in Normal and Fibrotic Human Lung Fibroblasts

We examined the level of the two isoforms of the TNFR in NF and FF. Flow cytometry was used to examine the level of TNFR1 and TNFR2 with specific antibodies for each isoform. The results, expressed as mean fluorescence intensity (MFI) of positive cells, revealed that the level of both isoforms was significantly lower in FF compared with NF. MFI of TNFR1 was 3.55 ± 0.12 for NF and 1.78 ± 0.35 for FF (P < 0.001). MFI of TNFR2 was 1.95 ± 0.27 for NF and 0.99 ± 0.16 for FF (P < 0.01). Values are represented as individual data points and mean of six different cell lines of NF and FF (Figure 3A). A representative series of histograms taken from the six different cell lines of NF and FF examined is displayed in Figure 3B.

View larger version (26K): [in this window] [in a new window]   Figure 3.   (A) Expression of TNFR1 and TNFR2 in NF (squares) and FF (triangles). Fibroblasts were fixed with paraformaldehyde and then incubated with goat antihuman TNFR1 and TNFR2. Cells were then labeled with antigoat IgG conjugated with FITC. Analysis at the flow cytometer was based on at least 10,000 forward and side scatter gated events for each specimen. Values are represented as individual data points and are the means of six different cell lines of NF and FF. (B) Representative flow cytometry histograms of TNFR1 and TNFR2 expression in NF and FF.

Effect of TNF- Treatment on Normal and Fibrotic Human Lung Fibroblasts

The ability of TNF- to induce PG production was markedly impaired in FF lines. Stimulation of FF with TNF- (20 ng/ml) produced 790 ± 90 pg/ml of PGE₂ versus 1,435 ± 144 pg/ml released by NF after 4 h of incubation (P < 0.05). Values represent mean ± SE of one cell line of NF and FF, each tested three times (Figure 4). This difference was related to a reduced ability of TNF- to induce expression of COX-2 in FF cell lines. Western blot analysis revealed that a 4-h treatment with 20 ng/ml of TNF- induced an increase of COX-2 expression in NF cell lines (3.75-fold increase) and a smaller induction in FF (2.35-fold increase) (Figure 5). In contrast, COX-1 expression was similar in NF and FF under basal conditions and was not modified by treatment with TNF- (Figure 5). The proliferation rate of NF, evaluated as percentage of MTT reduction, after treatment with TNF- (20 ng/ml/48 h) was 18.5 ± 2.7%, whereas the growth of FF cell lines was not modified by exposure to the cytokine (3 ± 2%) (P < 0.05) (Figure 6). Finally, the ability of TNF- to affect some functions related to collagen metabolism was also modified in FF. A 48-h exposure to TNF- caused a dramatic increase of the MMP-1 produced by NF (330 ± 91%) and a much smaller enhancement in FF (66 ± 21%) (P < 0.05) (Figure 7). On the contrary, the expression of the collagen receptor CD49 was stimulated by TNF- treatment (20 ng/ ml/48 h) in NF (42% over control levels) and much more so in FF (102% over control levels), the difference between NF and FF being significant (P < 0.05) (Figure 8).

View larger version (16K): [in this window] [in a new window]   Figure 4.   Amount of PGE2 produced by NF (open bars) and FF (closed bars) after 1 h and 4 h of TNF- treatment (20 ng/ml). Values represent the means ± SE of three different experiments.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Western blot analysis of COX-1 and COX-2 expression. After protein separation and transfer, the membrane was incubated with either goat anti-COX-2 or anti-COX-1 antibodies for 2 h before exposure to horseradish peroxidase-conjugated antigoat IgG. Proteins were visualized using a chemiluminescence detection system. Quantitation of COX-1 and COX-2 was carried out by scanning densitometry. A representative Western blot analysis and the related graphs are shown. Fibroblasts were exposed to TNF- (20 ng/ml) for 4 h. C = control.

View larger version (9K): [in this window] [in a new window]   Figure 6.   Proliferation rate of NF and FF after treatment with TNF- (20 ng/ml/48 h). Values are expressed as percentage of MTT reduction and are represented as individual data points and the means of four different cell lines of NF and FF.

View larger version (8K): [in this window] [in a new window]   Figure 7.   MMP-1 produced by NF and FF after TNF- treatment. Fibroblasts were maintained in serum-free medium for 18 h. Thereafter, TNF- (20 ng/ml) was added and cells incubated for 48 h. Values are expressed as percent of control. The data are represented as individual data points and are the means obtained from four cell lines of NF and FF.

View larger version (8K): [in this window] [in a new window]   Figure 8.   Expression of CD49 in NF and FF after TNF- treatment (20 ng/ml/48 h). Values are expressed as the percentage above control values. The data are represented as individual data points and are the means obtained from six cell lines of NF and FF.

	Discussion

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We have previously shown that NF, through the production of PGE₂, inhibit in vitro both TNF- messenger RNA accumulation and TNF- release by LPS-activated monocytes (12). We hypothesized that during the inflammatory process, activated monocytes modulate the inflammatory response and the subsequent reparative process through the release of cytokines such as TNF-. This cytokine can directly affect several fibroblast functions and also induce these cells to produce increased amounts of PGE₂, which can in turn exert a negative feedback on monocytes as well as an autocrine inhibition of fibroblast proliferation and function, leading to self-limitation of the inflammatory process. Chronic inflammation may represent the result of a failure in this homeostatic mechanism; a defective functioning in one of the cells involved could in fact result in an imbalance leading to disease.

To demonstrate this hypothesis in this study, we evaluated whether fibroblasts coming from diseased tissue exhibit different functional features compared with normal cells, with particular regard to the modulatory role supposed for NF in the context of the inflammatory process. Considering that PGE₂ is responsible for the inhibitory effect exerted by NF on monocyte TNF- production, we determined the content of PGE₂ in the conditioned medium produced by FF. Indeed, the four cell lines of FF that we tested spontaneously produce less PGE₂ than do NF, and as a consequence, they showed a reduced inhibitory effect on the production of TNF- by monocytes.

We also observed that the ability of TNF- to induce PGE₂ synthesis was impaired in fibrotic cell lines and was related to a reduction in the expression of COX-2. This was possibly due to the reduction of the expression of TNFR in fibrotic cell lines compared with normal cell lines (Figure 9). It has in fact been shown that TNFR1, one of the two types of TNFR, mediates TNF- biologic activity in terms of COX-2 expression and PGE₂ synthesis (18). Although the reduced TNFR1 expression may contribute to this decreased response, we cannot rule out an aspecific effect because an impairment of FF to increase COX-2 levels in response to interleukin 1 has also been observed (19). Based on these findings, FF have a reduced capacity to produce PGE₂ both spontaneously and after TNF- exposure. This may result in an uncontrolled release of TNF- from activated monocytes and, as an effect of the reduced expression of TNFRs, have a different impact of this cytokine on fibrotic cell activity. These changes may have important consequences on the development of inflammation and fibrosis, although the full involvement of TNF- in the induction of fibrosis is still controversial (20). However, TNF- is known to affect several fibroblast biologic activities such as proliferation as well as collagen and collagenase synthesis (21). Thus, we have analyzed the ability of TNF- to affect proliferation and some activities associated with collagen metabolism in NF and FF, examining both the levels of MMP-1 and the expression of collagen receptors because the destruction of collagen by enzymatic activity (26) and its phagocytosis upon binding to specific fibroblast receptors seem to be critical for collagen metabolism (27).

View larger version (26K): [in this window] [in a new window]   Figure 9.   Proposed scheme of the local control exerted by NF and FF on the inflammatory response. Inflammatory or fibrogenic stimuli activate monocytes to produce increased amounts of TNF-. By binding to TNFR, this cytokine stimulates COX-2 expression and PGE2 synthesis in NF. PGE2 in turn exerts a negative feedback on monocyte TNF- production as well as an autocrine inhibition of fibroblast proliferation and metabolic function leading to the self-limitation of the inflammatory response that characterizes the reparative process. The reduced expression of TNFR and the diminished activity of COX-2 in FF cause a reduced synthesis of PGE2 and, as a consequence, an impairment of their modulatory activity. These changes may lead to the self-perpetuation of the inflammatory process and to fibrosis.

Collagen phagocytosis is recognized as the preponderant mechanism of collagen degradation. In agreement with previous reports (30), treatment with TNF- increased the expression of ₂₁ integrin (CD49b), the principal receptor for collagen type I in NF, an effect that was maintained and even increased in FF. A reduced ability to phagocytize collagen has been reported in gingival fibroblasts exposed to TNF- (31), a phenomenon that has been related to the impairment of collagen turnover that may lead to fibrosis. Despite an increased expression of collagen receptors, TNF- may then contribute to the occurrence of fibrosis by disrupting collagen degradation at different steps. The higher expression of ₂₁ integrin in FF may then reflect the inability to generate appropriate collagen binding and internalization in response to TNF-. Interestingly, TNF- failed to increase MMP-1 levels in FF, an effect likely owing to the reduced expression of TNFR1 in these cells. Rekdal and colleagues (32) have in fact shown that TNF-induced collagenase production is mediated through TNFR1. The reduced capability of FF to produce MMP-1 may be relevant to the evolution of fibrosis because the degradation of extracellular matrix due to collagenase is often considered useful for the normal turnover of collagenous connective tissue (32), although some authors suggest that this event can make the infiltration into the tissue of inflammatory and structural cells easier (33).

The reduced expression of TNFR1 in FF may also explain the lack of growth inhibition in response to TNF- in these cell lines. In several cellular systems TNFR1 is known to mediate growth inhibition and death events, whereas TNFR2 is related to proliferative responses (34). A reduction of TNFR has already been reported in other conditions that reflect an inflammatory state, but an upregulation of TNFR1 in osteoarthritic synovial fibroblasts has been reported (18). We also cannot exclude that a shedding of both TNFRs may occur, though this phenomenon does not seem to play a crucial role in the reduction of TNFR expression in other systems (35).

The data we presented in this report support the hypothesis that under conditions of sustained lung injury and inflammation, subpopulations of fibroblasts with distinct phenotypic characteristics can emerge. This may affect the degradation and the metabolism of the extracellular matrix and most importantly may lead to a loss of their potentially important modulatory role. These events may have important consequences for the evolution of the inflammatory process toward chronicity. The concept that fibroblasts participate in shaping an inductive environment that might lead to a perpetuation of the inflammatory process raises the question of which events induce structural cells to express these activities. It is possible to hypothesize that inflammatory stimuli, such as cytokines and growth factors, may permanently affect fibroblasts, changing their phenotype; alternatively, we can also suppose that when inflammation goes beyond a certain point it causes the emergence, from a heterogeneous pool, of cells with specific features, such as the spontaneous release of proinflammatory cytokines and/or the reduced production of modulatory molecules such as PGs. Such an event would introduce into the system different patterns of cell-to-cell interaction, thereby increasing the likelihood for causing disease.