Increased subpopulations of CD16(+) and CD56(+) blood monocytes in patients with active Crohn's disease

BACKGROUND: Circulating monocytes may be subdivided according to the presence or absence of the Fcgamma receptor CD16 and the neural cell adhesion molecule CD56. Monocytes classified into these subpopulations are characterized by distinct phenotypic and functional features. We hypothesized that patients with active Crohn's disease differ in their peripheral monocyte subpopulations. METHODS: Using flow cytometry we investigated the expression of CD16 and CD56 on circulating monocytes in 11 patients with active Crohn's disease and 11 controls. These monocyte subpopulations were then analyzed for expression of the chemokine receptor fractalkine, CX(3)CR1, and the monocyte chemoattractant protein-1, CCR2. RESULTS: We found a median 3.7-fold increase in the number of CD16(+) monocytes related to the population with high expression of the pattern recognition receptor CD14 compared to that in the controls (P < 0.001). By studying the percentage of monocytes expressing CX(3)CR1, and their relative fluorescence intensity (RFI), we found significant differences, with both the highest percentage and the highest RFI in the CD14(low)CD16(+) subpopulation, whereas the CD14(high)CD16(+) subgroup represented an intermediate population. Inversely, CCR2 expression was highest in the populations with high expression of CD14, whereas the CD14(low)CD16(+) subpopulation showed the lowest percentage and the lowest RFI for CCR2. We found the percentage of CD14(+)CD56(+) monocytes in patients with active Crohn's disease to be increased 2.7 times compared to the controls (P = 0.011). CONCLUSIONS: These results show that subsets of peripheral monocytes with a more mature phenotype are expanded in patients with active Crohn's disease.     

Circulating monocytes in patients with diabetes mellitus, arterial disease, and increased CD14 expression

Low serum concentrations of high-density lipoprotein (HDL) cholesterol and elevated levels of acute-phase reactans are frequently found in patients with non-insulin-dependent diabetes mellitus (NIDDM) and cardiovascular disease. Changes in the phenotype of circulating monocytes have been reported with both of these circumstances in nondiabetic subjects. In the present study, we explored the possibility that similar changes may occur in circulating monocytes of patients with NIDDM and arterial disease. Two groups of subjects with NIDDM were studied: patients with cardiovascular disease (n = 25) were compared with a group without cardiovascular disease (n = 26); both groups were age- and sex-matched, had the same length of diabetes duration, and degree of glycemic control. Healthy nondiabetic volunteers of comparable age and sex (n = 35) formed the control group. There was no significant difference in the numbers of the CD14+/CD16+ monocyte subpopulations between the 3 groups. However, a significant graded increase of the mCD14 intensity expression values was observed among the groups, with the highest levels in patients with NIDDM patients and the lowest in nondiabetic subjects. The serum C-reactive protein concentrations were significantly higher in the group with arterial disease compared with those without arterial disease or healthy controls. In the group of patients as a whole, relative mCD14 intensity expression was significantly correlated with HDL cholesterol levels (inversely) and with serum concentrations of C-reactive protein. Serum HDL cholesterol levels and the C-reactive protein concentrations were also significantly correlated. We concluded that the increased mCD14 intensity expression on circulating monocytes may be an important contributor to the increased inflammatory response observed in patients with NIDDM and arterial disease, and eventually, to atherogenesis.     

Expression of CD40 and its ligand, CD40L, in intestinal lesions of Crohn's disease

OBJECTIVE: Selected mechanisms of the immune system participate in the development of inflammatory bowel disease. Recently, overexpression of the ligand for CD40 (CD40L), a lymphocyte costimulatory molecule, was shown to induce severe inflammatory bowel disease in transgenic mice. In the present study, we examined the expression of CD40 and CD40L on surgical specimens of ileum from 12 patients with Crohn's disease and 10 patients with diverticulitis. METHODS: Several CD40L+ cells were present in the affected tissue of patients with Crohn's disease, whereas few scattered CD40L+ cells were detected in sections of histologically normal ileum, resected distantly from the affected tissue, in patients with diverticulitis and in normal ileum portions obtained from colorectal cancer undergoing extensive surgery. The phenotype of CD40L+ cells was mainly CD4+. RESULTS: In patients with Crohn's disease, several CD40+ cells were detectable in the same areas of lymphocytes expressing CD40L, whereas in patients with diverticulitis, the number of CD40+ cells was significantly lower. Most of the CD40+ cells costained with CD20, thus showing to be B-lymphocytes, and only a few were CD14+ macrophages. Several von Willebrand-positive vessels were also positive for CD40. In addition, several infiltrating macrophages were found to express B7-1 and B7-2 molecules, the ligands of CD28 and CTLA-4, which cooperate with the CD40-CD40L pathway in lymphocyte activation. Staining of ileal lesions with anti-CTLA-4 antibodies resulted in detection of none or very few positive cells. In contrast, in patients with diverticulitis, an enhanced number of B7-1 and B7-2 and CTLA-4 was observed. CONCLUSION: The local accumulation of CD40L+ together with CD40+ cells within intestinal lesions of Crohn's disease suggests the involvement of this co-stimulatory pathway.     

Intestinal permeability is increased in a proportion of spouses of patients with Crohn's disease

OBJECTIVE: Increased small intestinal permeability has been found in patients with Crohn's disease and in a proportion of their healthy relatives. This may reflect a shared environment or shared genes. The finding of abnormal permeability in the healthy spouses of patients would favor an environmental cause for this observation. METHODS: The healthy spouses of patients with Crohn's disease attending three gastroenterology clinics were invited to participate. Eligible subjects consumed a 350-ml solution containing lactulose, mannitol, and sucrose before bedtime. All overnight urine was collected, assayed by high performance liquid chromatography, and the ratio of fractional excretion of lactulose to mannitol was calculated as an index of permeability. The results were compared with those of a previously determined control group. RESULTS: Sixty spouses completed the study. Increased permeability was present in eight (13.3%, 95% CI = 6.0-24.6%). The presence of increased permeability was not related to age, gender, duration of cohabitation, alcohol use, nonsteroidal anti-inflammatory drug use or to disease activity in the patient with Crohn's disease. There was a nonsignificant trend for abnormal permeability to occur in those spouses cohabiting with the patient with Crohn's disease at the time of disease diagnosis (p = 0.128). CONCLUSIONS: Small intestinal permeability is increased in a proportion of healthy spouses of patients with Crohn's disease. The presence of abnormal permeability studies in patients with Crohn's disease and a proportion of their healthy close contacts suggests that this phenomenon is caused by environmental factors.     

CD14 expression on monocytes and soluble CD14 plasma levels in correlation to the promotor polymorphism of the endotoxin receptor CD14 gene in patients with inactive Crohn's disease

BACKGROUND/AIMS: The association of the single nucleotide polymorphism in the promotor of the lipopolysaccharide receptor CD14 gene (T/C at position -159) with Crohn's disease has recently been demonstrated. This CD14 polymorphism is a potential predisposition factor responsible for inter-individual differing inflammatory reactions involving the CD14 receptor. We studied the correlation between the CD14 genotype (CC, CT, TT) and the membrane-bound CD14 monocyte expression and soluble CD14 in patients with inactive Crohn's disease. METHODOLOGY: In 23 patients and 29 healthy volunteers the membrane-bound CD14 density on unstimulated monocytes and soluble CD14 plasma levels were examined using quantitative flow cytometry and enzyme-linked immunosorbent assay. RESULTS: In normal controls membrane-bound CD14 monocyte density did not differ significantly between the genotypes CC, CT, or TT. In contrast, patients with inactive Crohn's disease and genotype TT showed a significantly lower membrane-bound CD14 density on monocytes compared to patients with genotype CC. Soluble CD14 plasma levels were significantly higher in patients with inactive Crohn's disease compared to the same genotype of healthy controls, but there was no significant difference between the genotypes CC, CT, and TT. CONCLUSIONS: Our data show that the membrane-bound CD14 monocyte expression and the soluble CD14 plasma levels in patients with inactive Crohn's disease completely differ from that in healthy individuals. In order to develop individualized therapy strategies further studies should be carried out to evaluate whether the TT genotype is associated with differences in the clinical course of Crohn's disease and in the response to antibacterial treatment.     

Treatment with infliximab is associated with increased markers of bone formation in patients with Crohn's disease

OBJECTIVE: Osteoporosis is a common complication of Crohn's disease (CD). Glucocorticoid use and detrimental effects of inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha) can lead to osteoporosis. The aim of this study was to assess the ability of treatment with the TNF-alpha antagonist infliximab to increase bone formation as measured by surrogate markers of bone turnover in patients with active CD. METHODS: Sera from 38 prospectively enrolled CD patients were examined for levels of bone alkaline phosphatase (BAP), N-telopeptide of type I collagen (NTX), immunoreactive parathyroid hormone (iPTH), calcium, and pro-inflammatory cytokines at baseline and 4 weeks following infliximab infusion. Crohn's Disease Activity Index (CDAI), Inflammatory Bowel Disease Questionnaire (IBDQ), and glucocorticoid dose also were collected. RESULTS: In this cohort, CDAI and IBDQ scores were significantly improved at week 4 (P<0.001). Infliximab therapy was associated with an increase in BAP, a marker of bone formation (P=0.010), whereas NTX, a marker of bone resorption, was not increased (P=0.801). Among 22 patients who were taking glucocorticoids, mean glucocorticoid dose decreased 36% (P<0.001; -7.9 mg). CONCLUSIONS: Treatment with infliximab was associated with increased markers of bone formation (BAP) without increasing bone resorption (NTX). This effect may be due to a beneficial effect of TNF-alpha blockade on bone turnover, a beneficial effect on CD activity resulting in decreased glucocorticoid dose, or both. Studies of longer duration are needed to assess the effect of infliximab on bone mineral density.     

Hemoperfusion with polymyxin B-immobilized fibers reduced the number of CD16+ CD14+ monocytes in patients with septic shock

BACKGROUND: CD16+ CD14+ monocytes dramatically increase in number in patients with severe infection. Hemoperfusion with PMX-F (direct hemoperfusion with polymyxin B immobilized fibers) has been reported to be a safe and effective treatment for patients with septic shock, although the molecular mechanism that accounts for its effectiveness is still unclear. The purpose of this study was to quantify the number of CD16+ CD14+ monocytes in patients with an intra-abdominal infection and to evaluate the effects of PMX-F treatment on clinical parameters and leukocyte surface antigen expression in these patients. MATERIALS AND METHODS: Seventeen septic patients who had an intra-abdominal infection were enrolled in this study; 7 of these patients received PMX-F treatment. Peripheral blood samples were obtained immediately after admission, and were also collected from the above 7 patients before, during, and immediately after their PMX-F treatment. The expression of CD14, CD16, and Toll-like receptor (TLR)-4 on these patients' monocytes was evaluated using flow cytometry. In addition, lipopolysaccharide (LPS)-induced production of TNF-alpha and IL-1beta by these cells was measured by ELISA. RESULTS: Monocytic expression of CD16 and TLR-4 was significantly greater in septic patients than in healthy controls, and their proportion of CD16+ CD14+ monocytes was similarly elevated. LPS-induced production of TNF-alpha and IL-1beta by peripheral blood mononuclear cells (PBMCs) of septic patients was significantly reduced compared to controls. Furthermore, there was a reduction in the proportion of CD16+ CD14+ monocytes during PMX-F treatment, and in the expression of TLR-4 on monocytes after PMX-F treatment. CONCLUSIONS: These results showed that the number of peripheral blood CD16+ CD14+ monocytes and monocytic TLR-4 expression were markedly increased, and the production of pro-inflammatory cytokines in response to LPS significantly reduced in patients with sepsis. PMX-F treatment was found to be effective in reducing the number of CD16+ CD14+ monocytes and in decreasing the monocytic expression of TLR-4 in patients with septic shock.     

Monocyte-derived dendritic cells of patients with coronary artery disease show an increased expression of costimulatory molecules CD40, CD80 and CD86 in vitro

BACKGROUND: Atherosclerosis is a disease triggered by diverse exogenous stimuli and sustained by chronic inflammatory processes. Dendritic cells (DCs) are key regulatory antigen-presenting cells and play a crucial role in regulating the adaptive and innate immune system in any chronic inflammatory process. DCs are present in atherosclerotic lesions in the areas of the highest T-cell density. So far, their role in atherosclerosis has not been fully elucidated. We investigated the phenotypic properties of DCs in patients with coronary artery disease (CAD) in comparison to healthy individuals. METHODS: Peripheral blood monocytes were isolated from 50 patients with CAD and 19 healthy individuals and differentiated over 9 days to immature and mature DCs. Analysis of the distribution of important stimulatory and costimulatory molecules on the surface of immature and mature DCs was performed by flow cytometry. RESULTS: We observed no changes between the groups concerning cell numbers or expression of CD1a or HLA-DR on DCs. Patients with CAD, however, showed a significant upregulation of the costimulatory molecules CD80, CD86 and CD40 as compared with healthy controls. Expression of CD40, CD80 and CD86 on DCs partly correlated with smoking, family history of CAD, as well as with C-reactive protein levels. High-density lipoprotein cholesterol was inversely associated with the expression of CD40 and CD80 on mature DCs (P<0.05). CONCLUSION: Upregulation of important costimulatory molecules on monocyte-derived DCs of CAD patients, is influenced multifactorially. Our results show notable differences between CAD patients and healthy individuals, possibly contributing to the pathophysiological processes in atherogenesis.     

An index of inflammatory activity in patients with Crohn's disease.

An objective and quantitative standard of inflammatory activity for patients with Crohn's disease was developed. This Activity Index (AI) was derived from data of 63 patients with Crohn's disease who had been submitted to a total of 85 clinical examinations. On the basis of 18 predictor variables three physicians gave an overall evaluation of the severity of inflammatory activity in each patient. Stepwise multiple regression analysis was used to investigate which combination of variables contributed most to the overall evaluation. The combination of the following nine variables gave a very good correlation (r = 0.95) with the overall evaluation: serum albumin, ESR, body weight related to length, abdominal mass, sex, temperature, stool consistency, bowel resection, and extraintestinal symptoms related to Crohn's disease. This combination of variables expressed in a score that was used as an activity index proved to be very useful in the assessment of disease activity and of the effect of therapy. Index values below 100 are associated with inactive disease, values between 100 and 150 can be regarded as indicating slight inflammatory activity, values between 150 and 210 as indicating moderate, and values above 210 as indicating severe-to-very-severe inflammatory activity.     

CD5+ B cells are decreased in peripheral blood of patients with Crohn's disease

B cells bearing the CD5 surface marker comprise a substantial minority of the circulating lymphocyte population in healthy individuals. These recently described cells have been implicated in T-independent humoral responses, immunoregulation, and autoimmunity. We undertook to enumerate circulating CD5+ B cells by three-color fluorescence activated flow cytometry in 28 patients with Crohn's disease (CD). None of the CD patients were using immunosuppressive medication. The CD patients were subdivided into "inactive" and "active" groups based upon their Crohn's disease activity index (CDAI). Thirty-two normal subjects served as a control population. The percentage of CD19+ B cells was significantly reduced in both active and inactive CD patients as compared with normal controls (P less than or equal to 0.01). CD5+ B cells were likewise found to be significantly decreased in both inactive and active CD patients (P less than or equal to 0.01) as compared with normal controls. The proportion of CD5+ B cells was significantly lower in the peripheral blood of active as compared with inactive CD patients (P less than or equal to 0.05). The finding that CD5+ B cells are reduced in CD may provide an important clue to immunological dysfunction in inflammatory bowel disease and merits further study.     

Mycophenolate mofetil in patients with Crohn's disease

Objective: Intolerance to azathioprine is a rare but important problem in treating chronically active Crohn's disease. We performed this study to evaluate mycophenolate mofetil as an alternative immunosuppressive therapy for patients with Crohn's disease who did not tolerate azathioprine.
Methods: Four patients with highly active perianal Crohn's disease and two patients with chronically active, steroid-dependent Crohn's disease were included. All patients consumed 2 g/day of mycophenolate mofetil for a median of 8 months (range, 6–12 months). Disease activity was measured by the Perianal Crohn's Disease Activity Index in patients with perianal disease and by the Crohn's Disease Activity Index in patients with chronically active Crohn's disease.
Results: Azathioprine-induced side effects disappeared after the drug was discontinued. All patients improved during treatment with mycophenolate mofetil, as shown by a remarkable reduction in the respective clinical scores. Five patients showed no side effects during treatment with mycophenolate mofetil. After 4 months' treatment one patient developed diarrhea that was probably not due to mycophenolate mofetil.
Conclusions: Mycophenolate mofetil could be an alternative therapy to azathioprine in patients with Crohn's disease.     

Narcotic use in patients with Crohn's disease

OBJECTIVES: Despite advances in treatment for Crohn's disease (CD), some patients suffer from chronic pain. We sought to characterize the prevalence of narcotic use and contributing factors in CD patients at a referral center. METHODS: A retrospective analysis of 291 CD patients followed over a 5-yr period was performed. Clinical status was evaluated with the Harvey-Bradshaw index (HBI) of disease activity and the short inflammatory bowel disease questionnaire (SIBDQ). RESULTS: Narcotic use was identified in 13.1% of patients. Narcotic users were more likely to be female, 72%versus 49% (p= 0.01), had higher rates of disability, 15.4%versus 3.6% (p= 0.001), and a longer duration of disease, 17.0 versus 12.9 yr (p= 0.03). In addition, they took more medications 6.97 versus 4.7 (p < 0.001) and had a higher prevalence of neuropsychiatric drug use, 37%versus 19% (p= 0.01). CD patients receiving narcotics had worse disease activity (HBI 9.1 vs 5.0, p < 0.001) and diminished quality of life (SIBDQ 44.2 vs 51.6 (p= 0.04)). However, logistic regression analysis found that active disease [HBI score of > or = 4 (OR 3.9)], polypharmacy [use of > or = 5 drugs (OR 5.5)], and smoking (OR 2.8) were associated with narcotic use. CONCLUSIONS: Narcotic use may be an indicator of more severe disease since it is associated with increased disease activity and decreased quality of life. Factors correlating with narcotic use include smoking and PP. Our data emphasize the need for further work to characterize chronic pain in CD patients.     

Corticosteroid-sparing treatments in patients with Crohn's disease

Conventional corticosteroid therapy effectively induces remission of Crohn's disease (CD) across a range of disease severity. However, alternative treatments are needed for patients with disease unresponsive to corticosteroids, patients requiring maintenance therapy (for which corticosteroids are ineffective), corticosteroid-dependent patients, and patients with corticosteroid-related toxicities. Thus, corticosteroid-sparing effects are an important clinical endpoint for treatments of CD. Budesonide offers comparable efficacy with less short-term toxicity than conventional corticosteroids (prednisone, prednisolone); this agent has also demonstrated short-term remission maintenance efficacy, while potentially enabling withdrawal of more toxic corticosteroids in corticosteroid-dependent patients. However, budesonide has not shown long-term maintenance benefit in clinical studies, and the risk for and implications of budesonide dependency need further evaluation. The immunomodulators, azathioprine and 6-mercaptopurine, are most effective for maintenance of remission in quiescent disease, but may be useful in conjunction with other therapies in inducing remission in active CD; methotrexate may be considered an alternative because of its efficacy in inducing and maintaining remission. In clinical trials, treatment with azathioprine/6-methotrexate has enabled corticosteroid withdrawal in 55% of patients, and methotrexate, in 39% of patients with corticosteroid-dependent CD, while maintaining clinical response. Monitoring for infrequent hematological or hepatic toxicity is recommended during use of these immunomodulators. Infliximab is effective for induction and maintenance of remission in patients with refractory CD participating in randomized placebo-controlled studies and, in open-label experience, has enabled corticosteroid withdrawal in approximately three quarters of patients. This biological agent is generally well tolerated. Infusion reactions are the most commonly occurring side effects; such reactions may require adjustment of infusion rate and/or treatment with an antihistamine or acetaminophen. The investigational biological agent CDP-571 has also shown corticosteroid-sparing efficacy in patients with CD. In conclusion, recent research has helped identify corticosteroid-sparing treatments that can provide benefit in patients with corticosteroid-dependent and/or corticosteroid-refractory CD or patients at risk for corticosteroid-induced toxicities.