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Keywords:

  • interleukin-12;
  • interleukin-23;
  • inflammatory bowel diseases

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Background: Interleukin (IL)-12p70 and IL-23 are key T helper-1 (TH1) cytokines that drive the inflammation seen in numerous models of intestinal inflammation. These molecules contain an identical p40 chain that is bound to a p35 chain in IL-12 and a p19 chain in IL-23, making both potentially susceptible to modulation by an anti-IL-12p40 monoclonal antibody (mAb).

Methods: In the present study, we sought to determine whether active inflammation in Crohn's disease (CD) is associated with the increased synthesis of both of these cytokines and whether patients treated with an anti-IL-12p40 mAb down-regulate IL-23 as well as IL-12p70 as previous reported.

Results: To this end we initially determined that IL-12p70 secretion by control and CD antigen-presenting cells (macrophages) in lamina propria mononuclear populations is optimized by stimulation with CD40L and interferon-γ. In subsequent studies using these stimulation conditions we found that patients with CD manifested both increased IL-12p70 and IL-23 secretion before anti-IL-12p40 mAb treatment and normal levels of secretion of these cytokines following cessation of treatment. Antigen-presenting cells in lamina propria mononuclear cells from ulcerative colitis patients, in contrast, produced only baseline levels of IL-23. Finally, we found that IL-23-induced T cell production of IL-17 and IL-6 are also greatly reduced after antibody treatment. The latter data are parallel to those from previous studies showing that anti-IL-12p40 down-regulates IFN-γ and tumor necrosis factor-α secretion.

Conclusions: We conclude that CD but not ulcerative colitis is associated with high levels of both IL-12p70 and IL-23 secretion as well as the secretion of downstream effector cytokines, and that this cytokine production is down-regulated following administration of IL-12p40 mAb.

In recent years it has become increasingly apparent that regardless of its underlying genetic basis, the inflammation of Crohn's disease (CD) ultimately depends on the development of a T helper-1 (TH1) cytokine response.1 What is meant by a TH1 response has recently undergone some redefinition, however. Whereas for many years the TH1 response has been synonymous with the production of interleukin (IL)-12p70, a heterodimeric cytokine composed of a p40 and a p35 chain, it is now apparent that a TH1 response can also be driven by IL-23, a related cytokine composed of a p40 chain (which it shares with IL-12p70) and a p19 chain that is unique to IL-23.2,3 Evidence for the dual character of the TH1 response comes mainly from studies of murine models of inflammation such as experimental allergic encephalomyelitis (EAE) and collagen-induced arthritis, in which it has been shown using mice lacking one or the other of these cytokines that the induced inflammation depends to an equal or greater extent on an IL-23-driven response than on an IL-12p70-driven response.4,5 This appears to be related to the fact that although the IL-12p70 response results in T cells engaged in the production of interferon-γ (IFN-γ), the IL-23 response results in T cells that produce both IL-17 and IL-6.2,3 Whereas there are no published reports of the role of IL-23 in murine models of gut inflammation, the studies of other murine models of TH1 inflammation raise the question of the role of IL-23 in CD. In a study of CD relevant to this issue, Schmidt et al have shown that IL-23 measured by real-time reverse transcriptase-polymerase chain reaction is increased in the inflamed mucosa of CD patients and to a lesser extent in patients with ulcerative colitis (UC).6 Thus, there is already little question that the TH1 response in CD will also be a complex response having both IL-12p70 and IL-23 components.

In the present study, we show that IL-23 protein secretion is indeed elevated in patients with CD as is T cell production of IL-17 and IL-6. Moreover, we show that patients subjected to therapy with anti-IL-12p40 mAb manifest a dramatic decrease in IL-23 and IL-23-induced cytokines. These studies show that an anti-IL-12p40 antibody affects both IL-12p70 and IL-23 and therefore may be effective in ameliorating disease regardless of which TH1 cytokine is the more important proinflammatory cytokine.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients

Colectomy specimens for the study of IL-12p70 secretion were obtained from 40 surgical patients admitted for bowel resection. The CD group consisted of 4 men and 11 women, ranging from 19 to 66 years of age, and the UC group consisted of 2 men and 8 women, ranging from 23 to 55 years of age. The diagnosis for each patient was made using clinical parameters, radiographic studies, and histological criteria. At the time of resection, 7 patients of the CD group were receiving corticosteroids, 1 patient was receiving oral aminosalicylic acid (ASA) preparation, and 7 patients were taking no medications. In the UC group, 4 patients were receiving corticosteroids, 1 patient was taking oral ASA preparation, and 5 were taking no medications. The control group consisted of colonic specimens from 15 patients admitted for therapeutic bowel resection for malignant (adenocarcinoma; N = 13) and nonmalignant (diverticulosis; N = 2). There were 2 men and 13 women in the control group ranging in age from 36 to 92 years.

Patients studied for cytokine secretion before and after anti-IL-12p40 monoclonal antibody (mAb) administration consisted of 8 CD patients. They ranged in age from 32 to 66 years. Five patients had disease limited to the colon and 3 patients had ileocolonic disease. At the time of the study, 4 patients were receiving oral ASA preparations, 1 patient was receiving corticosteroids, and 1 patient was taking 6-mercaptopurine, whereas 4 patients were receiving no medications. The Crohn's Disease Activity Index (CDAI) score for each patient is reported in Figure 1. One patient in this group (patient 2) was randomly assigned to receive placebo but inadvertently received a single dose of anti-IL-12p40 mAb. Because this patient did not receive a full course of anti-IL-12p40 mAb therapy these patient cytokine results were not reported within the context of this study. The control group consisted of colonic biopsy specimens collected from 3 patients with irritable bowel syndrome. The administration of anti-IL-12p40 mAb was carried out as previously reported.7 In brief, patients were randomized blindly to receive placebo, 1 mg/kg, or 3 mg/kg of anti-IL-12p40 mAb. Patients in regimen 1 were administered 7 weekly subcutaneous doses with a 4-week safety review period between the first and second doses; patients in regimen 2 were administered 7 uninterrupted weekly subcutaneous doses. The collection of lamina propria samples was approved by the appropriate ethical committee and institutional review boards.

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Figure FIGURE 1. Rates of clinical response and remission among patients with active CD receiving 7 subcutaneous injections of anti-IL-12p40 mAb or placebo either in an interrupted (A) or uninterrupted (B) dosage. Shaded areas mark 7-day periods after injections.

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Lamina Propria Mononuclear Cell Isolation

Lamina propria mononuclear cells (LPMCs) were isolated form freshly resected mucosa tissue using a dithiothreitol (DTT)-EDTA-collagenase method described previously.8 LPMC were isolated from biopsy material using a modification of the aforementioned method. In brief, biopsy tissue was washed in Hanks' balanced salt solution (HBSS) free of calcium and magnesium (HBSS-CMF Hyclone, Cramlington, Neb). They were then incubated in 10 mL of 1X phosphate-buffered saline (PBS) containing 1.5 mg DTT (Sigma Chemical Co., St. Louis, Mo) for 5 min at room temperature on a rotating agitator. After 2 washes in HBSS-CMF, the biopsy material was chopped into a fine mesh and then incubated in 15 mL of HBSS-CMF containing 0.75 mmol/L EDTA for 15 min at room temperature to remove epithelial cells. After these washes, the mucosal fragments were washed twice with HBSS. The tissue specimens were then incubated for 1 hour at 37°C in a humid 5% CO2 atm in medium (RPMI-1640 + 10 mmol/L HEPES buffer, 2 mmol/L glutamine, 10% heat-inactivated fetal calf serum (FCS; Hyclone), and antibiotics [penicillin 100 U/mL; streptomycin 100 μg/mL; and fungizone 0.25 μg/mL]) containing 25 U/mL collagenase V (Sigma). After incubation, the supernatant was collected and washed twice in HBSS-CMF and LPMC were subjected to centrifugation through a 40%/70% Percoll gradient (Sigma). LPMC were collected at the 40%/70% interface. The lamina propria macrophage population was then enriched from the resultant population by negative selection techniques using mAbs attached to immunomagnetic beads (Dynal Corporation, Oslo, Norway). In some control experiments, peripheral blood macrophages from healthy volunteers were prepared using the same procedure. In brief, cell populations were suspended at 2 × 107 cells/mL in calcium-free PBS with 1% FCS to which a 1:350 dilution of ascites fluid containing antibodies to OKT4 (anti-CD4; ATCC, Rockville, Md), OKT8 (anti-CD8; ATCC), THB5 (anti-CD21; ATCC), 10F7 (anti-glycophorin), and 3G8 (anti-CD16) was added. The cells were incubated at 4°C for 30 min, washed twice, and resuspended in coating medium (PBS with 1% FCS). The antibody-coated cell populations were then removed by incubation with immunomagnetic beads coated with anti-murine immunoglobulin G antibody obtained from Dynal Corporation. The resultant macrophage cell population contained >90% pure population, as assessed by flow-cytometric analysis.

Cell Cultures

Isolated lamina propria macrophages were resuspended at a final concentration of 1 × 106 cells/mL in complete medium consisting of RPMI-1640 + 10 mmol/L HEPES buffer, 2 mmol/L glutamine, and antibiotics supplemented with 10% FCS (Gemini Bioproducts, Woodland, Calif.) and cultured for 48 hours in 48-well plates (Costar, Corning Inc, Corning, NY) in humid 5% CO2 atm. For evaluation of IL-17 and IL-6 production from T cells, LPMC cells were cultured in the presence of soluble anti-CD28 mAb + soluble anti-CD2 mAbs (T112 and T113). The anti-CD2 (T112 and T113) was used at a 1:1000 final dilution and the anti-CD28 mAb was used at a final concentration of 1 μg/mL. To some of the LPMC or LP macrophage samples, 1 ng/mL bacterial lipopolysaccharide (LPS; Sigma), 0.07 vol/vol% Staphylococcus aureus Cowan's antigen (SAC; Sigma), 10 μg/mL CD40L (Amgen Corp, Seattle, Wash), or 100 U/mL of recombinant IFN-γ (R & D Systems, Minneapolis, Minn) was added. At the end of the 48-hour culture period, supernatants were collected and stored at −80°C until tested for cytokine content. Samples were assayed for IL-12p70, IL-17, and IL-6 in duplicate by the Luminex bead technology. Concentrations were determined with a Luminex100 cytometer (Luminex Corp, Austin, Tex) using BioPlex Manager software (Bio-Rad Laboratories, Hercules, Calif) and Linco Multiplex Cytokine Kits (Linco Research, St. Charles, Mo) according to the manufacturer's instructions. In supernatants stimulated for IL-23 production, supernatants were measured using a commercially available kit from Bender Medsystems (Vienna, Austria) per the manufacturer's instructions. Optical densities were measured on a Dynatech MR 5000 enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

Statistical Analysis

Statistical analysis of the data was performed using Student's t test. Changes in mean cytokine secretion before and after treatment were evaluated by the paired t test analysis using PRISM 4 statistical software (Graph Pad software, San Diego, Calif).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

IL-12p70 Production by Purified Antigen- presenting Cells Obtained From CD and UC Surgical Resection Specimens

In initial studies we sought to determine the optimal conditions for the expression of IL-12 family cytokines by antigen-presenting cells (APCs) in the lamina propria. To this end, we isolated LPMCs from CD, UC, and control patient colectomy specimens and then obtained purified APC populations (macrophages) from the LPMCs by negative immunomagnetic selection (see Methods). We then cultured the purified APCs in the presence of a variety of stimuli and measured IL-12p70 secreted into the culture medium by ELISA. As shown in Figure 2A, whereas LP APCs isolated from control patients produced low amounts of IL-12p70 when stimulated with SAC or LPS as compared with their peripheral blood counterparts, the same LP APCs produced substantial amounts of IL-12p70 when stimulated with CD40L and even greater amounts of this cytokine when stimulated with CD40L and IFN-γ. With this information in hand, we compared IL-12p70 production by LP APCs isolated from CD, UC, and control tissue. As shown in Figure 2B, LP APCs from CD patients stimulated with CD40L or CD40L plus IFN-γ produced statistically increased amounts of IL-12p70 compared with APCs from controls (P < 0.01, CD40L + IFN-γ CD as compared with controls). In contrast, LP APCs from UC patients produced substantially less IL-12p70 than APCs from controls when stimulated with CD40L + IFN-γ (P < 0.02). Taken together, these studies show that IL-12p70 production is greatly enhanced with stimulation of APCs with CD40L + IFN-γ as compared with other commonly used APC stimuli such as SAC or LPS alone, and that CD LP APCs produce greatly increased amounts of IL-12p70 after such stimulation.

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Figure FIGURE 2. A, Production of IL-12p70 protein secretion by peripheral blood macrophages and LP macrophages after indicated stimulation pathways. B, Production of IL-12p70 protein secretion by LP macrophages from patients with CD, UC, and control patients after indicated stimulation pathways.

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IL-12p70 and IL-23 Production by LPMCs Obtained From Biopsy Specimens of CD Patients Before and After Treatment With Anti-IL-12p40 mAb Treatment

The above results of optimizing stimulation for LPMC APC (macrophage) IL-12p70 production enabled the determination of both IL-12p70 and IL-23 secretion by APC (macrophages) isolated from biopsy specimens of CD patients undergoing a study of the safety and efficacy of anti-IL12p40 treatment. As reported previously,7 in these studies it was shown that the administration of humanized anti-IL-12 mAb directed against the p40 subunit of IL-12 leads to a high clinical response and remission rate. In particular, 75% of patients that received 7 uninterrupted weekly 3 mg/kg subcutaneous doses achieved a clinical response, as defined by a drop of >100 points in the CDAI score and 50% of patients achieved clinical remission, as defined by a CDAI score of <150. As shown in Figure 1, these results were mirrored in the subgroup of patients studied at the National Institutes of Health who were subjected to endoscopic biopsy for the collection of tissue for cytokine analysis. A majority of the patients in this subgroup who were administered 7 weekly uninterrupted 3 mg/kg doses also achieved a long-lasting clinical response and/or remission.

Patients receiving anti-IL-12p40 treatment underwent colonoscopy before the initiation of treatment and within 48 hours after cessation of treatment. LPMCs were isolated from the biopsy specimens (see Methods) and cultured with CD40L IFN-γ and SAC for 48 hours (the addition of SAC to culture stimulation conditions containing CD40L + IFN-γ was observed to further increase IL-12p70 levels by 10%), after which culture supernatants were collected and assayed by ELISA for the presence of IL-12p70 or IL-23. Moreover, other aliquots of LPMCs were cultured with anti-CD2/anti-CD28 for the same time period and culture supernatants were harvested and assayed by ELISA for the presence of IL-17 and IL-6. Additional cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α) were also measured and have been reported previously.7 It should be noted that cytokine production by LPMCs rather than purified APCs or T cells were measured in these studies, and therefore the cytokine production was subject to sampling variations arising from differences in the relative number of APCs and T cells in each set of biopsies.

As shown in Figure 3A and reported previously,7 IL-12p70 secretion by CD LPMCs obtained from pretreatment biopsies was elevated compared with secretion by control LPMCs (153.1 ± 51.1 versus 50.1 ± 27.5 pg/mL; control N = 3; P < 0.05) and LPMC obtained from posttreatment biopsies (IL-12p70 pretreatment values: 153.1 ± 53.1 pg/mL versus IL-12p70 posttreatment values: 2.8 ± 2.6 pg/mL significantly different at P < 0.03, N = 8). Furthermore, in studies of responses of individual patients (also shown in Figure 3A), 7 of the 8 patients studied exhibited decreases in IL-12p70 secretion and, interestingly, the 1 patient (patient 8) whose LPMC secretion of IL-12p70 did not decline significantly did not experience a sustained clinical response (see Figure 1).

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Figure FIGURE 3. Cytokine secretion by mononuclear cells of the colonic LP for (A) IL-12p70 and (B) IL-23 from patients with CD before and after treatment with anti-IL-12p40 mAb. Symbols indicate patient number from Fig. 1.

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In further studies we determined whether CD patients exhibited parallel changes in LPMC IL-23 secretion. As shown in Figure 3B, we found that pretreatment LPMC IL-23 secretion was also increased in a majority of the patients studied compared with both control LPMC secretion (175 ± 63 versus 29 ± 21 pg/mL; P < 0.03) and posttreatment CD LPMC secretion (175 ± 63 versus 25 ± 12.0 pg/mL; P < 0.03). The 1 patient (patient 8) mentioned above whose cells did not manifest a decrease in IL-12p70 also did not exhibit a posttreatment decrease in IL-23 secretion. Furthermore, LPMCs of UC patients produced less IL-23 than pretreatment LPMC of CD patients (44.8 ± 14 versus 175 ± 63 pg/mL; P < 0.02) and indeed, the level of UC IL-23 secretion (N = 10) was not significantly different from control LPMC secretion (N = 3) (44.8 ± 14 versus 29 ± 21 pg/mL; P > 0.05).

IL-17 and IL-6 Production by LPMCs Obtained From Biopsy Specimens of CD Patients Before and After Treatment With Anti-IL-12p40 mAb Treatment

In a final series of studies, we measured LPMC production of IL-17 and IL-6, 2 cytokines that are induced by IL-23 but not by IL-12p70. For this purpose, we stimulated the LPMCs with anti-CD2/anti-CD28, a polyclonal T cell-stimulating antibody combination (see Methods). As shown in Figure 4A and 4B, pretreatment T cell production of IL-17 and IL-6 was increased as compared with posttreatment LPMC T cell secretion. IL-17 pretreatment secretion levels were 141 ± 60 pg/mL versus posttreatment secretion levels of 12.1 ± 3.0 pg/mL, whereas P < 0.03; and IL-6 pretreatment was 1355 ± 501 pg/mL versus posttreatment secretion levels of 54 ± 19 pg/mL, P < 0.02). Once again, LPMCs from patient 8, the only nonresponding patient, did not exbihit a decrease in IL-17/IL-6 secretion in parallel to the lack of IL-23 decrease.

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Figure FIGURE 4. Cytokine secretion by mononuclear cells of the colonic LP for (A) IL-17 and (B) IL-6 from patients with CD before and after treatment with anti-IL-12p40 mAb. Symbols indicate patient number from Fig. 1.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present study, we presented evidence that CD is associated with a multifaceted TH1 response characterized by not only an increased LPMC production of IL-12p70 but also the production of a more recently discovered TH1 cytokine, IL-23 and its downstream T cell cytokines, IL-17 and IL-6. It should be noted that CD is also associated with the production of the IL-12p70-induced cytokine IFN-γ as well as a cytokine induced by both IL-12p70 and IL-23, TNF-α. In initial studies we determined the ability of isolated LPMC APCs (macrophages) from patients with both CD and UC to produce IL-12p70 in response to various stimuli, recognizing that such information would be essential to further studies of the production of this cytokine during an evaluation of the effect of anti-IL-12p40 mAb on patients with CD. Here we found that such APCs produce relatively low levels of IL-12p70 when stimulated with conventional APC stimuli such as LPS and SAC when compared with peripheral APCs that produce relatively high levels of IL-12p70 after such stimulation. This may account for the findings in a previous study that showed that although LPMC APCs from CD patients stimulated with either LPS or SAC alone manifested increased IL-12p70 production when compared with controls, the IL-12p70 levels actually measured were quite modest (i.e., in the 10-30 pg/mL range).9 One possible explanation for this altered activation pattern relates to the fact that LP APCs are by their location more exposed to Toll-like receptor (TLR) ligands than peripheral APCs and that such exposure may result in a type of tolerance (unresponsiveness) that affects responses to various TLR ligands.10-12 Such tolerance would certainly affect responses to LPS, which depend on TLR4, but it would also affect SAC responses because the latter also stimulates cells via TLRs.13 In contrast to these findings, we found that responses of LP macrophages to CD40L (plus IFN-γ) was preserved or even increased compared with those of peripheral macrophages. Thus, LP APCs retain the ability to respond to an activated T cell stimulus (CD40L) even when they respond poorly to a TLR stimulant. A further illustration of the relevance of T cell stimulation to IL-12 production by LP macrophages is inherent in the requirement of IFN-γ for optimal IL-12 production, because the latter suggests that initial T cell stimulation of IL-12p70 production by intestinal APCs is rapidly reinforced within a milieu that contains T cells undergoing TH1 differentiation and IFN-γ production.

In summary of these initial studies, we found that stimulation of LPMC APCs from CD patients with CD40L + IFN-γ led to greatly enhanced IL-12p70 responses, whereas stimulation of APCs from UC patients with these stimuli led to diminished IL-12 responses. These studies thus provide further data that support the concept that CD is a TH1 inflammation, whereas UC is not.

Having optimized the in vitro stimulation conditions, we measured IL-12p70 and IL-23 responses of LPMCs extracted from biopsies of tissues taken from active CD lesions before and after anti-IL-12p40 mAb administration. As reported previously, such treatment led to a striking down-regulation of IL-12p70 responses and, in parallel studies of anti-CD2/anti-CD28-stimulated T cells, of the downstream IL-12p70 cytokine IFN-γ.7 We now report that such therapy also down-regulated LP APC production of IL-23 and its downstream T cell cytokines, IL-17, and IL-6. These results establish 2 essential features of the TH1 inflammation coexisting with CD. First, long-standing active inflammation in CD is associated with an expanded complement of TH1 cytokines and therefore does not conform to a sequential model of TH1 cytokine production in which IL-12p70 is supplanted by IL-23. Second, existing anti-IL-12 p40 antibody has a dual capacity to address both major TH1 components.

The importance of TH1 cytokine secretion to the pathophysiology of CD lies in the fact that increased IL-12p70/IL-23 production and its ensuing TH1 T cell cytokine response is directly responsible for the pathological changes that are characteristic of the disease. This was nicely illustrated in earlier studies of human mucosal tissue injury studied in explants of fetal intestinal tissue.14 In these studies it was shown that activation of T cells in the explants with anti-CD3 alone elicits little IFN-γ production and subsequent tissue injury whereas, in contrast, activation with IL-12 and anti-CD3 resulted in a massive increase in the amount of both IFN-γ and TNF-α and was associated with significant tissue injury. Thus, T cell stimulation in the absence of a TH1-directing cytokine originating from an APC does not result in sufficient cytokine production to support tissue injury. Further studies using fetal explants indicated that the tissue injury was secondary to the induction of increased levels of activated matrix metalloproteins such as interstitial collagenase and stromelysin-1 and the latter, in turn, were dependent on the secretion of IL-12, IFN-γ, and TNF-α.14 Support for this cytokine dependence came from the fact that neutralization of IL-12 led to a direct decrease in tissue injury as well as a decreased induction of activated metalloproteins. These studies, taken together, provide a clear link between IL-12p70 responses and tissue injury and thus establish that this component of the TH1 response in CD cannot be discounted as a major factor in the pathophysiology of the disease.

The latter conclusion in no way precludes the parallel role of IL-23 as a major proinflammatory TH1 cytokine involved in tissue injury. Strong evidence for the latter possibility comes from recent studies of TH1 T cell-mediated inflammation characterizing EAEs (a murine model of multiple sclerosis) in which it has been shown that mice lacking the ability to produce IL-23 because they cannot synthesize the IL-23 p19 chain (i.e., p19−/− mice produced by gene targeting) were resistant to the development of EAE and CNS inflammation despite the abundance of IFN-γ-producing T cells within the CNS lesions, presumably arising from an intact IL-12p70 response.15 IL-17-producing T cells were undetectable in these animals, suggesting that IL-23 was essential for the development of auto-antigen-specific T cells producing IL-17 and that the latter, in turn, was responsible for the CNS pathology.4,15 IFN-γ secretion in this model may in fact have an anti-inflammatory effect because it was found that IL-23 secretion is enhanced in the absence of IL-12p70 and IFN-γ secretion.15 In related studies, it has been shown that p19−/−-deficient mice also display a resistance to collagen-induced arthritis, another TH1 T cell-mediated inflammation, in this case affecting the joints.5 Although mice with collagen-induced arthritis manifest increased IL-17 levels, increased IL-12p70 levels are also evident and thus the overexpression of IL-12p70 downstream effector cytokines (IFN-γ and/or TNF-α) may also participate in tissue injury in this model.

The above data relating to the role for IL-17 as an important mediator of various autoimmune disease processes are supported by numerous studies on the various activities of this cytokine. Among these are studies showing that IL-17 alone or in combination with TNF-α stimulates endothelial cells and macrophages to produce several factors that regulate the influx and subsequent expansion of inflammatory cells (particularly granulocytes) at local sites of inflammation. These factors include adhesion molecules such as intracellular adhesion molecules, CXC chemokines such as CXCL8 (IL-8 ligand) and CXCL6, and growth factors such as granulocyte colony-stimulating factor.16 In addition, IL-17 also acts as a more direct proinflammatory mediator through its capacity to stimulate macrophage production of inflammatory cytokines such as IL-1, IL-6, and TNF-α2,3 and to stimulate epithelial cells and fibroblasts to secrete neutrophil chemoattractant factor (IL-8) and neutrophils to release cationic proteins and acid proteases; the latter factors, alone or in combination with IL-6 and TNF-α, are directly cytotoxic to tissue through their ability to degrade extracellular matrix components and to increase intestinal permeability.17,18 Finally, IL-17 has been shown to inhibit posttranscriptional modification of IL-6 and IL-8 mRNA that would lead to degradation of the mRNA encoding these cytokines, thereby increasing the net excretion of these cytokines.19,20 Taken together, these data establish IL-17 as an effector cytokine with a multifaceted and potent effect on cytokine and chemokine responses associated with inflammation. Nevertheless, it is premature to conclude that IL-17 is solely responsible for tissue injury in mucosal inflammations in general and CD in particular given the known effects of IFN-γ discussed above. On the contrary, it seems more likely at this stage that both the IL-12p70 and IL-23 components of the TH1 response have important roles in these inflammations.

In summary, our data clearly suggest that mucosal APCs secrete increased amounts of both IL-12p70 and IL-23 in CD and this leads to the production of T cell cytokines that account for the tissue injury characteristic of the disease. Thus, administration of an anti-IL-12p40 mAb that recognizes both of these cytokines provides a therapeutic approach that addresses both arms of the dysregulated TH1 process underlying CD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr Scott Fritz, SAIC Corp, Frederick, Maryland, for the kind gift of hybridomas 1057 and 3G8; Dr Ellis Reinhertz, Dana Farber Cancer Institute, Boston, for the anti-CD2 mAb pair (T112 and T113); and Prof Carl June, Cancer Center, University of Pennsylvania, Philadelphia, for the anti-CD28 mAb (clone 9·3).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References