Defective macrophage handling of Escherichia coli in Crohn's disease.

BACKGROUND AND AIM: Escherichia coli can be isolated from lamina propria macrophages in Crohn's disease (CD), and their intramacrophage persistence may provide a stimulus for inflammation. To further determine the contributions of macrophage dysfunction and E. coli pathogenicity to this, we aimed to compare in vitro functioning of macrophages from patients with CD and healthy controls (HC) in response to infection with CD-derived adherent-invasive E. coli (AIEC) and less pathogenic E. coli strains. METHODS: Monocyte-derived macrophages were cultured from patients with CD and HC. Intramacrophage survival of E. coli strains (CD-derived adherent-invasive [AI] and non-AI strains and laboratory strain K-12) was compared. Macrophage cytokine release (tumor necrosis factor alpha [TNFα], interleukin [IL]-23, IL-8 and IL-10) and monocyte phagoctyosis and respiratory burst function were measured after E. coli infection. For CD patients, laboratory data were correlated with clinical phenotype, use of immunomodulation, and CD risk alleles (NOD2, IL-23R, ATG16L1 and IRGM). RESULTS: Attenuated TNFα and IL-23 release from CD macrophages was found after infection with all E. coli strains. There was prolonged survival of CD-derived AIEC, CD-derived non-AIEC and E. coli K-12 in macrophages from CD patients compared to within those from HC. No abnormality of monocyte phagocytosis or respiratory burst function was detected in CD. Macrophage dysfunction in CD was not influenced by phenotype, use of immunomodulation or genotype. CONCLUSIONS: CD macrophage responses to infection with E. coli are deficient, regardless of clinical phenotype, CD genotype or E. coli pathogenicity. This suggests host immunodeficiency is an important contributor to intramacrophage E. coli persistence in CD.

Lithium counteracts histamine suppression of human T cell mitogenesis.

Human T cell proliferative responses to concanavalin A (conA) were suppressed by approximately 50% by histamine (100 microM). In contrast, LiCl (1 or 3 mM) potentiated T cell responses by about 50%, but 10 mM LiCl had no significant effect on T cell proliferation. Histamine suppression was not significantly affected by the presence of potentiating concentrations of LiCl, whereas 10 mM LiCl completely abrogated histamine suppression.

Prostaglandin E2-mediated enhancement of human plasma cell differentiation.

Addition of prostaglandin E2 (PGE2) to blood mononuclear cell cultures containing pokeweed mitogen (PWM) enhances plasma cell (PC) differentiation measured by intracytoplasmic immunoglobulin 7 days later. T-cell mitogenesis to concanavalin A is inhibited using the same concentrations of PGE2. PGE2 failed to enhance the PC differentiation of lymphocytes from patients with systemic lupus erythematosus (SLE). Indomethacin, on the other hand, either had no effect or suppressed PC differentiation. The data is discussed in terms of the effect of PGE2 on human suppressor T-cell function.

Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from mast cells from various locations.

Nedocromil sodium and sodium cromoglycate inhibited histamine release from rat peritoneal mast cells. Tachyphylaxis was observed with both drugs. The 2 compounds were extremely selective in their action, being less active against peritoneal mast cells from the hamster and completely ineffective against mast cells from the mouse. Human basophil leucocytes, tissue mast cells of the guinea-pig and rat intestinal mast cells were also unresponsive. Both drugs inhibited immunological histamine release from human pulmonary mast cells obtained by bronchoalveolar lavage (BAL) and, less effectively, from lung parenchyma. Nedocromil sodium was about 1 order of magnitude more potent than sodium cromoglycate in each case. Tachyphylaxis was observed with the dispersed lung, but not with the cells obtained by BAL, and the degree of inhibition varied inversely with the magnitude of the secretory response. The possible clinical significance of these observations is discussed.

Secretory and accessory cell functions of the alveolar macrophage.

We have attempted to address the requirements necessary for alveolar macrophage accessory cell function. We have also examined the in vitro and in vivo factors that must be taken into account when interpreting results from experimental studies. Differences in phenotypic expression by rat alveolar pleural and peritoneal macrophages are noted, as well as the differing expression of major histocompatibility complex (MHC) class II molecules. Furthermore, alveolar macrophages, harvested from rat lung, do not express the interleukin (IL)-1 cytokines, and lipopolysaccharide (LPS) treatment of quiescent cells (after 24-hr in vitro culture) induces low levels of expression of IL-1 alpha and IL-1 beta. Short-term inhalation of refractory ceramic fibers, however, results in markedly increased IL-1 beta expression after stimulation with LPS. We suggest that, in vivo, IL-1 beta may be involved in the initial recruitment and activation of inflammatory cells rather than in induction of immune responses. We also postulate, based on recent published evidence, that alveolar macrophages activate the dendritic cells within the respiratory epithelium. Thus alveolar macrophages would release cytokines critical for the activation of dendritic cells during the afferent limb of the immune response, and they would respond to products of sensitized T-cells such as interferon-gamma and IL-4 to interact with T-helper cells in an antigen-specific MHC-restricted manner during the efferent limb of the response.

In vitro exposure effects of cyclosporin A and bis(tri-n-butyltin)oxide on lymphocyte proliferation, cytokine (receptor) mRNA expression, and cell surface marker expression in rat thymocytes and splenocytes.

Rat thymocytes and splenocytes were exposed in vitro to the model compounds Cyclosporin A (CsA), an immunosuppressive drug, and bis(tri-n-butyltin)oxide (TBTO), an immunotoxic environmental contaminant. The lymphocyte transformation test (LTT), cytokine (receptor) mRNA expression (RT-PCR and dot blot hybridisation), and flow cytometry were evaluated as assays for in vitro immunotoxicity, at dose levels that did not show effects on viability, this being the aim of the study. LTT and RT-PCR proved useful assays. Lymphocyte transformation was suppressed by both compounds, while IL-2 mRNA expression was suppressed by CsA but not by TBTO, and both compounds suppressed IL-2R mRNA expression in splenocytes but not in thymocytes. Furthermore, the data obtained suggest that antiproliferative effects may be more relevant than apoptosis induction for TBTO induced thymus atrophy.

Distinct microbial populations exist in the mucosa-associated microbiota of sub-groups of irritable bowel syndrome.

BACKGROUND: There is increasing evidence to support a role for the gastrointestinal microbiota in the etiology of irritable bowel syndrome (IBS). Given the evidence of an inflammatory component to IBS, the mucosa-associated microbiota potentially play a key role in its pathogenesis. The objectives were to compare the mucosa-associated microbiota between patients with diarrhea predominant IBS (IBS-D), constipation predominant IBS (IBS-C) and controls using fluorescent in situ hybridization and to correlate specific bacteria groups with individual IBS symptoms. METHODS: Forty-seven patients with IBS (27 IBS-D and 20 IBS-C) and 26 healthy controls were recruited to the study. Snap-frozen rectal biopsies were taken at colonoscopy and bacterial quantification performed by hybridizing frozen sections with bacterial-group specific oligonucleotide probes. KEY RESULTS: Patients with IBS had significantly greater numbers of total mucosa-associated bacteria per mm of rectal epithelium than controls [median 218 (IQR - 209) vs 128 (121) P = 0.007], and this was chiefly comprised of bacteroides IBS [69 (67) vs 14 (41) P = 0.001] and Eubacterium rectale-Clostridium coccoides [52 (58) vs 25 (35) P = 0.03]. Analysis of IBS sub-groups demonstrated that bifidobacteria were lower in the IBS-D group than in the IBS-C group and controls [24 (32) vs 54 (88) vs 32 (35) P = 0.011]. Finally, amongst patients with IBS, the maximum number of stools per day negatively correlated with the number of mucosa-associated bifidobacteria (P < 0.001) and lactobacilli (P = 0.002). CONCLUSIONS & INFERENCES: The mucosa-associated microbiota in patients with IBS is significantly different from healthy controls with increases in bacteroides and clostridia and a reduction in bifidobacteria in patients with IBS-D.

Sparing effect of cyclophosphamide (NSC-26271) pretreatment on animals lethally treated with gamma-irradiation.

Cyclophosphamide (CP) can protect mice from gamma-irradiation-induced lethality, the timing of the dose of CP in relation to the irradiation being the critical factor. The most sparing effect is achieved when CP is injected 3 days before irradiation. The protection is mediated by enhanced hemopoietic recovery seen in animals given the most favorable combination ofCP and gamma-irradiation. This rapid recovery cannot be explained by the reduced sensitivity of hemopoietic stem cells after CP treatment to subsequent irradiation although survival of a few radioresistant stem cells cannot be ruled out. Stimulus to repopulate by nonspecific depletion of the bone marrow is not the explanation either, since CP itself appears to play an important role in this effect. The role of the spleen is not critical in this phenomenon and attempts have failed to demonstrate the presence of a humoral factor which can restore bone marrow depleted by gamma-irradiation in the serum of animals treated with CP. CP given 3 days before irradiation appears to result in a purely additive effect against malignant tissue in mice, providing a useful differential kill on tumor tissue at the same time that it provides the maximum protection of normal tissue.

Enhanced post-irradiation recovery of the haemopoietic system in animals pretreated with a variety of cytotoxic agents.

It is known that pretreatment of mice with bacterial endotoxin and certain stathmokinetic agents between 1 and 3 days prior to exposure to ioninzing radiation reduce radiation lethality. In this communication it is shown that pretreatment with cytosine arabinoside, methotrexate, nortestosterone and chlorambucil reduces radiation (1000 rad) induced lethality. This reduction can be ascribed to enhanced regeneration of the haemopoietic system in pretreated animals and not to increased survival of colony-forming cells (CFU) in these animals. Regeneration of CFUs was underway within 24 hr after 900 rad in the pretreated mice but did not start until day 3 in mice treated with gamma radiation only. Two agents, namely radiation itself (either 75 or 150 rad) and busulphan (10 mg/kg) did not reduce the lethal effects of subsequent gamma irradiation nor enhance the regeneration of CFUs, even though radiation, like the protective cytosine arabinoside, induces early CFUs proliferation. The administration of nucleoside precursors of DNA enhanced regrowth of haemopoietic stem cells to an extent comparable with that of the most effective pretreatment, cytosine arabinoside. It is postulated that drugs like cytosine arabinoside operate by causing cell death, providing a source of DNA that con enhance the regrowth of surviving stem cells in the bone marrow.

Effect of prostaglandins and cyclic nucleotides on growth and immunoglobulin secretion of two IgE myeloma cell lines.

The effect of various mediators on the growth and secretion of IgE by two human myeloma cell lines derived originally from the same tumour was tested. It was found that the growth of U266 was unaffected by PGE2, but IgE secretion was blocked. PGF2 alpha, whilst inhibiting growth, had little effect on IgE secretion. With the second cell line, U266 BL, it was found that none of the agents tested could modulate the secretion of IgE, though cell growth was blocked by PGE2. Prostaglandins act by modulating cyclic nucleotides, the E series increasing the level of cAMP and the F series causing a rise in cGMP. Our findings with prostaglandins could be mimicked by the relevant cyclic nucleotide. Possible explanations for these differences are discussed.

The role of prostaglandins in the modulation of histamine suppression of mitogen responses in atopic and normal subjects.

We have shown that lymphocytes from atopics are more sensitive to histamine-induced suppression of mitogen transformation than are cells from normal subjects. Recent reports have suggested that histamine suppression may act via prostaglandin synthesis. We have investigated this and found that indomethacin partially reversed the histamine suppression seen in atopics, but not that in normals. This effect was seen even when the direct enhancing effect of indomethacin was taken into account. The direct effect of indomethacin on mitogen-induced transformation was similar in both normal and atopic cultures, which suggests that these results are not due to the presence of PG producing cells in atopic cultures, but rather that histamine modulates the function of these cells. It was found that a specific H2 agonist, but not an H1 agonist could partially mimic the effect of histamine this system.

Reduced lethality in mice receiving a combined dose of cyclophosphamide and busulphan.

Animals treated with a sufficiently high dose of busulphan die about 14 days later from bone marrow failure. A single, appropriately timed injection of cyclophosphamide can save these mice. The nature of this protection is shown to be the cyclophosphamide induced elaboration of a humoral factor which stimulates haemopoietic recovery.

Lack of immune deficiency in sarcoidosis: compartmentalisation of the immune response.

The original findings of peripheral anergy in sarcoidosis led to the conclusion that sarcoidosis was a disease associated with immune deficiency, but patients with sarcoidosis do not appear to suffer from repeated infections suggestive of immune suppression. With the technique of bronchoalveolar lavage it is now possible to examine the local immune response within the lung, the most commonly affected organ in sarcoidosis. In this study three different indices of cell mediated immunity (lymphocyte transformation, interleukin-2 production, and interleukin-1 production) have been examined by comparison of cells recovered by lavage with those collected from peripheral blood. It was found that in vitro anergy was confined to peripheral blood cells, where all three markers of the immune response used in this study was impaired in the 12 patients with sarcoidosis group when compared with results in the 12 controls, with the most depressed responses seen in those patients classified as having active disease (lymphocyte proliferation 45% (SD 17%); interleukin-2 production 44% (13%), and interleukin-1 production 31% (10%) of control levels). By contrast, T lymphocytes recovered from the lungs of patients with sarcoidosis showed a greater response than did those from controls in terms of lymphocyte transformation and interleukin-2 production; these differences were greatest in those with active disease (lymphocyte proliferation 209% (27%) and interleukin-2 production 202% (19%) of control levels). Interleukin-1 production by cells of the monocyte lineage recovered from the lung gave similar results to those of the control and sarcoid groups. It is concluded that the anergy seen in the peripheral blood compartment possibly reflects redistribution of T lymphocytes rather than a generalised immune deficiency.

Methionine-enkephalin stimulates in vitro proliferation of human peripheral lymphocytes via delta-opioid receptors.

The influence of methionine-enkephalin (Met-Enk) on in vitro proliferation of human peripheral lymphocytes was investigated. Met-Enk, in the concentration range 10(-12) to 10(-4) M enhanced the proliferative response to suboptimal concentrations of concanavalin A of human peripheral lymphocytes and to cells in the absence of mitogen. The response to Met-Enk in the absence of mitogen was not influenced by the presence of fetal calf serum: similar levels of enhancement were seen in cultures supplemented with 10% autologous serum. Enhancement of proliferation was blocked in a concentration-dependent manner by both naloxone and the delta specific antagonist ICI-174864. The sensitivity to the antagonistic influence of ICI-174864 suggests strongly that the stimulatory influence of Met-Enk on human lymphocyte proliferation in the absence of mitogen is mediated via the delta-opioid receptor.

Local increase in interleukin-1-like activity following UVB irradiation of human skin in vivo.

Using an in vivo skin chamber method, we demonstrated increased release of interleukin-1 (IL-1)-like activity at the site of irradiation with 3 times the minimal erythema dose of ultraviolet B (UVB). IL-1-like activity was estimated using the mouse thymocyte amplification assay. UVB-augmented release of IL-1-like activity peaked 1 h after irradiation and levels returned to baseline by 2 h. Release of IL-1-like activity from human skin after exposure to UV radiation may account for some of the local and systemic features of the sunburn response.

Nasal response to allergen and hyperosmolar challenge.

Rhinitis causes both clinical and social discomfort to patients, and in clinical practice is often underdiagnosed. We have examined a simple method for the assessment of a positive nasal provocation test to help in the diagnosis of rhinitis. In patients with histories suggestive of house dust mite (HDM) sensitivity and positive skin-prick tests or specific IgE to Dermatophagoides pteronyssinus, there was a fall in nasal inspiratory peak flow (NIPF) following nasal challenge with allergen. This was not seen in control subjects or in pollen-sensitive patients when challenged with house dust mite. Frequency of sneezing and degree of rhinorrhoea increased in these patients following challenge, and based on these findings we propose a simplified scoring system for the diagnosis of allergic rhinitis. We examined non-specific nasal reactivity using hyperosmolar solutions as a challenge system and found that allergic subjects responded with a fall in NIPF, although the clinical response was not identical to that seen with allergen. Control subjects did not respond to hyperosmolar challenge.

T gamma cells in sarcoidosis: E rosetting monocytes suppress lymphocyte transformation.

Increased suppressor T gamma lymphocytes have been described in sarcoidosis. We have shown that a proportion of these cells are esterase-positive, phagocytic, adherent to plastic and stain with an anti-monocyte serum. Removal of these cells or the addition of indomethacin increases the lymphocyte transformation to Con A. Transformation was still reduced in spite of preincubation with plastic and the addition of indomethacin suggesting that a further abnormality exists. Thus, within the increased number of T gamma cells there exists a population of activated monocytes which rosette with sheep red blood cells and could therefore be mistaken for T cells.

Effect of high-dose melphalan on marrow and intestinal epithelium in mice pretreated with cyclophosphamide.

The lethal effect of high-dose melphalan in mice could be offset by pretreatment with cyclophosphamide, cytosine arabinoside or low-dose melphalan. The reason for improved survival is unclear. Althoug animals given high-dose melphalan died with symptoms of gut death, in only one instance, that with low-dose melphalan itself, did pretreatment protect the intestinal epithelium as measured by the microcolony assay. A small enhancement in the recovery of the haemopoietic tissue in pretreated animals was noted, although this on its own is unlikely to explain the phenomenon. Experiments in tumour-bearing mice showed that pretreatment with cyclophosphamide did not reduce the toxicity of melphalan to the Lewis lung carcinoma.

Stimulation of human peripheral lymphocytes by methionine enkephalin and delta-selective opioid analogues.

The opioid peptide methionine enkephalin was shown to stimulate human peripheral lymphocyte proliferation in vitro in the absence of mitogen. A study of the time course of stimulation revealed a maximum proliferative response, measured by [3H]thymidine incorporation, after 4-5 days incubation. The kinetics of the response are similar to those for in vitro T cell responses to antigen rather than via polyclonal activation through lectin or anti-CD3 triggering, suggesting a physiological basis for the phenomenon. The stimulatory influence was blocked by the delta-selective antagonist ICI-174864, suggesting the mediation of classical opioid delta receptors. The opioid receptor specificity is further demonstrated using delta- and mu-selective agonists. The delta-selective agonists DSLET and DPDPE stimulate proliferation, whereas the mu-selective agonist DAGON was without effect.

Increased natural killer cell activity in sarcoidosis.

We have studied NK cell activity in 32 patients with sarcoidosis and in 29 control subjects. Cytotoxicity was increased in both active and inactive sarcoids, the lytic activity of sarcoid peripheral blood mononuclear cells (PBMC) being approximately 50% higher than that of control subjects. The number of lymphocytes bearing Fc receptors for IgG (FcR-IgG) was correlated with NK cell activity, both parameters being increased in the sarcoid group. Sarcoid NK cell activity was reduced to control levels after depletion of plastic adherent cells, whilst inhibition of cytotoxicity by PGE2 was increased using non-adherent sarcoid PBMC. Plastic depletion had no effect on NK cell activity or its inhibition by PGE2 using PBMC from control subjects. The results demonstrate the presence of a weakly adherent, PGE2 insensitive NK cell in sarcoid PBMC preparations.