Stefano Fais

Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease

BACKGROUND & AIMS Transmural perivascular mononuclear cell infiltrates are a feature of Crohns disease. The aim of this study was a molecular characterization of the mechanisms leading to the formation of these infiltrates. METHODS Endothelial cell and leukocyte expression of the adhesion molecules directing leukocyte transendothelial migration were studied in situ by immunohistochemical analysis of 10 samples from patients with Crohns disease and 10 samples from normal controls. Double-staining methods were used to characterize the cells forming the infiltrates. RESULTS CD11a+ and L-selectin-positive mononuclear cells seemed to be the major component of perivascular infiltrates. The vast majority of these cells were CD68+, CD31+ monocytes/macrophages surrounded by CD3+, L-selectin-positive, CD31+, CD45RA+, and/or CD45RO+ T lymphocytes. T lymphocytes within the vessels expressed both CD45RA and CD45RO markers. Endothelial cells were intercellular adhesion molecule 1 positive and mostly CD34+. Strong adhesion between L-selectin-positive and CD11a+ intravascular mononuclear cells and CD34+ and intercellular adhesion molecule 1-positive endothelial cells were observed. CONCLUSIONS Data indicate that peripheral mononuclear cells are actively recruited in the submucosa of Crohns disease tissue; endothelial cells express adhesion molecules highly permissive for transendothelial migration of monocytes and both naive and memory T cells contributing to infiltrates generation; and close membrane contact between migrated macrophages and naive T cells leads to the T-cell transition from naive to memory phenotype within Crohns disease.

Recombinant gp120 induces IL-10 in resting peripheral blood mononuclear cells; Correlation with the induction of other cytokines

Immutiological abnotmalities present in HIV‐1‐infected individuals often reflect an imbalance of cytokine production. The HIV‐I gpl20 has the ability to induce a number of cytokines, and to enhance immunoglobulin release by normal peripheral blood mononuclear cells (PBMC) in vitro, in the absence of IL‐2 production and of lymphoproliferation. This study provides evidence that gpl20 is a potent IL‐10 inducer in normal PBMC cultures. The pattern of other cytokines induced by gp 120 includes interferon‐alpha (IFN‐a) and IFN‐γ, tumour necrosis factor‐alpha (TNF‐α), IL‐6, IL‐Iα2 and IL‐β, and not IL‐2 and IL‐4. These findings further define the pattern of cytokine release induced by gpl20 on human resting PBMC. Furthermore, the present findings roughly parallel those observed both in the sera of patients and in the mononuclear cells from HIV+ individuals early after infection, suggesting that gpl20 could be a good candidate as one of the agents responsible for cytokine dysregulation observed in HIV‐1‐infected individuals.

Vasoactive intestinal polypeptide modulates the in vitro immunoglobulin a production by intestinal lamina propria lymphocytes

BACKGROUND/AIMS Vasoactive intestinal polypeptide (VIP) modulates the immunoglobulin (Ig) synthesis in the peripheral blood of humans and in various lymphoid organs of other species. Because VIP receptors have been shown on lamina propria mononuclear cells (LPMC), the effects of graded doses of VIP on the in vitro Ig production by normal human LPMC was investigated. METHODS Colonic LPMC were cultured with or without graded doses of VIP (1 pmol/L-1 mumol/L). The concentrations of Ig A, G, and M in the 12 days supernatants were quantitated by an enzyme-linked immunosorbent assay, and the effects of VIP were characterized by limiting dilution analysis (LDA). RESULTS The addition of VIP was associated with a significant increase in the production of IgA, whereas IgG levels were significantly reduced. Both these effects were restricted to LPMC. LDA showed that the IgA-enhancing effect was associated with an increase in the number of IgA-producing precursor cells and with variation in the regulatory phenomena involved in IgA production. CONCLUSIONS In humans, a major function of the mucosal immune system (i.e., the synthesis of IgA) is modulated by VIP. Data suggest that VIP may either act as an indirect switch factor or increase the clone size of pre-committed cells.

Effects of somatostatin on human intestinal lamina propria lymphocytes : modulation of lymphocyte activation

We have investigated the effect of somatostatin (SOM) on the mitogen-induced activation of lamina propria mononuclear cells isolated from the human intestinal mucosa (LPMNC) and of the autologous peripheral blood lymphocytes (PBMNC). The occurrence of specific SOM receptors and their biological characteristics were also investigated. The counts of interleukin-2 receptor (IL-2R)-positive cells after mitogen stimulation were significantly lower in the presence of SOM. This effect of SOM appeared to be dose dependent, with SOM concentrations ranging between 1 pM and 1 microM. The amount of SOM required for the 50% inhibition of this expression was 1000 times lower in the LPMNC population than in the PBMNC. Binding studies showed that human LPMNC bear specific receptors for SOM and demonstrated that the affinity of these receptors was 1000 times higher than that of the SOM receptors present on the PBMNC (Kd 2.1 +/- 0.34 nM vs. 910 +/- 46 nM, respectively). The inhibitory effect of SOM on the proliferative response appeared to be restricted to PBMNC, with a maximal inhibition at 1 nM SOM, while LPMNC proliferative response was poorly affected. SOM inhibited the in vitro immunoglobulin production of both PBMNC and LPMNC over a wide range of concentrations, with a maximal inhibition at 1 nM. At this concentration the effect of SOM on IgA was more pronounced in the PBMNC than in the LPMNC. Our results lend support to the concept that in humans SOM plays a role in the modulation of the immune response at the level of the intestinal mucosa where cell-to-cell interactions between SOM releasing nerve fibers and cells and the immune system occur.

Modulation of human natural killer activity by vasoactive intestinal peptide (VIP) family. VIP, glucagon and GHRF specifically inhibit NK activity

Vasoactive intestinal polypeptide (VIP) is a neuropeptide, which also modulates some immune functions. Natural killer (NK) cell activity was already found to be diminished by VIP. In the present paper we report that VIP is able to decrease NK cell activity of human large granular lymphocytes (LGL), showing maximal inhibition at doses ranging from 10(-8) to 10(-6) M. Some neuropeptides, belonging to the VIP family (secretin, glucagon, peptide histidine isoleucine, PHI and human growth hormone releasing factor, GHRF), were also tested. Among these peptides, secretin and PHI were shown to be uneffective on NK cell activity whereas glucagon and GHRF were inhibitory. The D50 of GHRF was similar to that of VIP (10(-9) M), the D50 of glucagon was 10(-8) M. A recently synthesized VIP-antagonist (4Cl-D-Phe6-Leu17) was then used to assess its ability to reverse the VIP-mediated inhibition of NK activity. The antagonist was able to completely reverse the inhibitory effect of VIP on NK activity. The VIP-antagonist was also able to reverse the inhibitory effect of glucagon and GHRF, even though to a lesser extent than for VIP. Our data provide a new physiological observation regarding the functional activity of LGL, supporting the presence of a receptor for VIP on human LGL with NK activity.

Inhibitory effect of somatostatin-14 and some analogues on human natural killer cell activity

The effect of somatostatin-14 (SST) and the SST analogues SMS and RC160 on human natural killer (NK) activity mediated by large granular lymphocytes (LGL), as well as on IL-2- and/or anti-CD16 monoclonal antibody (mAb)-induced activation of these cells, was investigated. The NK activity of LGL was studied by the release of 51Cr by the erythroleukemia-derived cell line K562, whereas 51Cr release by the P815 murine mastocytoma-derived cell line, for which lysis was redirected by the use of an anti-CD16 mAb, was used to study the cytolytic potential of these cells. IL-2 was used at the final concentration of 100 IU and was incubated overnight with LGL. SST and the analogues, added to these systems at final doses ranging from 10(-12) to 10(-5) M, were inhibitory of the NK cell activity to K562, with a dose-response curve starting from 10(-8) M and reaching a significant level at 10(-6) M. On the contrary, no effect was observed on the redirected killing assay to P815 and on the IL-2-induced activation of NK cells. These results provide additional evidence for the immunomodulatory action of somatostatin.

Effects of High In Vivo Levels of Vasoactive Intestinal Polypeptide on Function of Circulating Lymphocytes in Humans

To examine the possible in vivo significance of the immunomodulatory effects of vasoactive intestinal polypeptide described in vitro, several parameters of peripheral blood lymphocyte function were studied in a patient with a pancreatic endocrine tumor and high circulating levels of vasoactive intestinal polypeptide. There was no imbalance of the circulating lymphocyte subpopulations, and the in vitro responses of the patients lymphocytes to mitogens were normal. However, there was an increased number (32%) of peripheral lymphocytes expressing interleukin 2 receptor. Serum immunoglobulin M levels were higher than in controls, and the patients lymphocytes exhibited a spontaneous in vitro immunoglobulin M production higher than normal. Comparable increases in both interleukin 2 receptor expression and immunoglobulin M production were induced in vitro in normal peripheral lymphocyte cultures by the addition of vasoactive intestinal polypeptide concentrations similar to that detected in the patients plasma. These findings indicate that a modulatory effect of vasoactive intestinal polypeptide on lymphocyte activation and immunoglobulin synthesis may be operating in vivo. They also suggest that vasoactive intestinal polypeptide does not mediate major defects in peripheral blood lymphocyte function in vivo.

A Limiting-Dilution Analysis of Activated Circulating B Cells in Crohn's Disease

In the present study the spontaneousin vitro production of immunoglobulins G, A, and M by peripheral mononuclear cells was evaluated, in patients with Crohns disease, in relation to the state of B-cell activation and further characterized by limiting-dilution analysis. A total of 25 patients with Crohns disease and 10 healthy controls was studied. The proportion of the transferrin receptor-bearing cells in the B7+ subset was higher in active Crohns disease patients than in either those with quiescent disease or controls. There was a significant rise in thein vitro IgG, IgM, and IgA production in patients with untreated active Crohns disease compared to either those with untreated quiescent disease or controls. When patients were followed up from the active phase to clinical remission, a significant decrease in the production of IgG and IgM was observed. IgA levels also showed a decrease, although not reaching statistical significance. When the Ig production was analyzed by limiting dilution, no difference was observed between patients and controls in terms of either precursor frequency of Ig-producing cells or patterns of frequency distribution. In both patients and controls a biphasic limiting-dilution profile was observed. This study shows that a significant B-cell activation is present in Crohns disease patients, which is accompanied by an increase in immunoglobulin production. This study also indicates that in Crohns disease the increased immunoglobulin production is related to an augmented B-cell clone size rather than to an increased precursor frequency.

Peripheral and intestinal lymphocyte activation after in vitro exposure to cow's milk antigens in normal subjects and in patients with Crohn's disease

We studied in Crohns disease and controls the in vitro activation of either peripheral (PBMNC) or intestinal (LPMNC) mononuclear cells in response to the cows milk antigen beta-lactoglobulin (Blg). The activation of mononuclear cells was investigated by analyzing the kinetics of the transferrin receptor (T9 antigen) and interleukin 2 receptor (Tac antigen) expression. In both controls and Crohns disease patients Blg (1 microgram/ml) induced a significant (P less than 0.01) increase of T9 and Tac expression by both PBMNC and LPMNC cultured for 3 days. After Blg exposure the counts of T9- and Tac-bearing cells were significantly (P less than 0.05) higher in PBMNC cultures from healthy controls than in those from patients with Crohns disease. In both groups peripheral T-enriched cell suspensions did not express T9 and Tac when stimulated with Blg but were restored to express either antigen by the addition of 10% autologous adherent cells. In LPMNC cultures from patients with Crohns disease the Tac expression after 3 days of Blg stimulation was significantly (P less than 0.05) higher than in the autologous PBMNC. Data from this study indicate that in both controls and patients with Crohns disease lymphocytes sensitized to Blg occur in the circulation as well as in the gut lamina propria. Our data also suggest that in Crohns disease an increased proportion of lymphocytes sensitized to Blg are recruited from the circulation into the site of the intestinal lesion.

Persistent In Vivo Activation and Transient Anergy to TCR/CD3 Stimulation of Normal Human Intestinal Lymphocytes

Intestinal lymphocytes represent a unique lymphoid compartment heavily and constantly exposed to antigens. Continuous antigen challenge however, does not normally result in an inflamatory tissue damage, suggesting that cell activation mechanisms are tightly controlled in gut mucosal lymphocytes. Normal intestinal lymphocytes have been found in fact to express in vivo some degree of phenotypic activation, yet little if any evidence of in vivo proliferation is detectable. Accordingly, TCR/CD3 mediated in vitro proliferative signals are consistently inefficient. This peculiar behavior renders the intestinal immune system an ideal model to unravel in vivo operating mechanisms of suppression and tolerance induction.1