Mast cell degranulation induced by type 1 fimbriated Escherichia coli in mice.

The strategic location of mast cells at the host-environment interface and their ability to release potent mediators of inflammation have suggested that these cells may play a pivotal role in host defense against bacterial infection. The ability of the opportunistic pathogen, Escherichia coli, to induce degranulation of mast cells obtained from the mouse peritoneum was investigated. We determined that unlike a mutant derivative deficient in the FimH subunit of the fimbriae or nonfimbriated E. coli, type 1 fimbriated E. coli induced mast cell degranulation in vitro. The magnitude of mast cell degranulation was directly proportional to the number of adherent bacteria on the cell surface in the initial period of the interaction. Using a mouse model of bacterial peritonitis, we demonstrated mast cell degranulation and histamine release by type 1 fimbriated bacteria in vivo. Furthermore, beads coated with FimH but not with FimA, the major subunit of type 1 fimbriae, evoked mast cell release of histamine in vivo in amounts comparable to that elicited by type 1 fimbriated E. coli. These studies reveal that mast cells can be degranulated by interaction with type 1 fimbriated E. coli and that FimH, the mannose-binding component of the fimbriae, is a potent mast cell stimulant.

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection.

The FimH subunit of type 1-fimbriated Escherichia coli has been implicated as an important determinant of bacterial adherence and colonization of the urinary tract. Here, we sought to localize the functionally important domain(s) within the FimH molecule and to determine if antibodies against this domain would block adherence of type 1-fimbriated E. coli to the bladder mucosa in situ and in vivo in an established mouse model of cystitis. We generated translational fusion proteins of disparate regions of the FimH molecule with an affinity tag MalE, and tested each of the fusion products in vitro for functional activity. The minimum region responsible for binding mouse bladder epithelial cells and a soluble mannoprotein, horseradish peroxidase, was contained within residues 1-100 of the FimH molecule. We validated and extended these findings by demonstrating that antibodies directed at the putative binding region of FimH or at synthetic peptides corresponding to epitopes within the binding domain could specifically block type 1 fimbriae-mediated bacterial adherence to bladder epithelial cells in situ and yeast cells in vitro. Next, we compared the ability of mice passively immunized intraperitoneally with antisera raised against residues 1-25 and 253-264 of FimH or 1-13 of FimA to resist bladder colonization in vivo after intravesicular challenge with type 1-fimbriated E. coli. Only the antibody directed at the putative binding region of FimH (anti- s-FimH1-25) significantly reduced E. coli bladder infections in the experimental mouse model of urinary tract infections. Similar results were obtained when the mice were actively immunized with synthetic peptides corresponding to residues 1-25 and 253-264 of FimH or 1-13 of FimA. The mechanism of protection was attributed, at least in part, to inhibition of bacterial adherence to the bladder surface by s-FimH1-25-specific antibody molecules that had filtered through the kidneys into the urine. The level of FimH antibodies entering the bladder from the circulatory system of the immunized mice was found to be markedly enhanced upon bacterial challenge. The potential broad spectrum activity of the protective FimH antibody was indicated from its serologic cross-reactivity with various urinary tract bacterial isolates bearing type 1 fimbriae. These findings could be relevant in the design of an efficacious and broadly reactive FimH vaccine against urinary tract infections.

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

Clinical implications of mast cell-bacteria interaction.

Mast cells are traditionally known for mediating allergic reactions. In addition, these cells have been implicated in the pathogenesis of a variety of clinical conditions such as atopic and contact dermatitis, bullous pemphigoid, fibrotic lung disease, neurofibromatosis, psoriasis, scleroderma, rheumatoid arthritis, interstitial cystitis, ulcerative colitis, and Crohn's disease, but their role in host defense was an enigma until recently. Owing to the strategic location of mast cells at the host environment interface, their role in bacterial infections has been studied by a number of investigators. Latest reports show that mast cells have an ability to modulate the host's innate immune response to infectious agents. This review discusses the clinical implications of mast cell-bacteria interactions.

Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis.

Stimulated mast cells release a variety of chemotactic factors such as tumor necrosis factor alpha (TNF-alpha) and leukotriene B4. Recent studies have shown that mast cell-derived TNF-alpha plays a critical role in host defense against Gram negative bacterial infections by the recruitment of neutrophils to the sites of infection. In the present study, we sought to investigate if mast cells release leukotriene (LT) B4 in response to bacteria and, if so, to establish its in vivo relevance. We show that mast cells release significant amounts of LTB4 and LTC4 in response to exposure to FimH-expressing type 1 fimbriated Escherichia coli in vitro. To test the functional significance of mast cell-derived LTs during an E. coli infection in vivo, we examined the effect of a LT-synthesis inhibitor, A-63162, on bacterial clearance and neutrophil influx in an infectious peritonitis model in mast cell-deficient mice (WBB6F1-W/WV) and their normal congenic control (WBB6F1-+/+) mice. Our results show that a treatment with A-63162 reduced neutrophil influx and bacterial clearance in the peritoneal cavities of mast cell-sufficient but not -deficient mice. Thus, mast cell-derived LTs contribute to host defense by mediating early neutrophil influx and bacterial clearance at sites of infection.

Histamine in human epidermal cells is induced by ultraviolet light injury.

Human epidermal cell cultures were examined to determine whether they were capable of histamine release. Results of these studies indicated that keratinocytes contain and release significant amounts of histamine. In the skin of some individuals, histamine content was induced after ultraviolet B light injury, and 40% of subjects demonstrated high basal histamine levels. Mass spectrometric analysis of cell supernatants showed that the histamine was released into the extracellular environment. Such release may contribute to common itching or intensify the inflammatory response in vivo.

Mast cells as modulators of host defense in the lung.

Mast cells display a distinct spatial distribution in the lung where they are found preferentially in intraepithelial locations or in deeper tissue around blood vessels, bronchioles and mucus secreting glands. Yet the physiological role of these granule-laden cells is unknown. There are now intriguing signs that their distinctive distribution together with their intrinsic capacity to release large amounts of inflammatory mediators serve a critical role in immune surveillance. Mast cells have now been shown to be capable of recognizing and aggressively reacting to a wide range of bacteria. The mast cell responses involve ingesting and killing of adherent bacteria, in a manner not unlike that of traditional phagocytic cells. Concomitant with this endocytic activity, a large variety of potent inflammatory mediators are released by the mast cell. One such mast cell-derived mediator, TNF-alpha, was recently shown to be a critical signal for initiating neutrophil influx to sites of bacterial infection in the lung as well as the peritoneum of mice. This capacity of mast cells to recruit neutrophils, together with its recently reported participation in processing and presenting bacterial antigens to immune cells and in mediating proliferation of epithelial cells and mucosal mucus secretion, indicate that mast cells have an extraordinary ability to modulate the innate as well as adaptive immune responses to infectious microorganisms.

Histamine as an autocrine regulator of leukemic cell proliferation.

We examined leukemic lymphocyte precursors from ALL patients as well as immortalized ALL cell lines for cytoplasmic histamine expression. The histamine levels ranged from 10.8 pg/10(6) cells to 82.2 pg/10(6) cells in ALL cell lines (N=4) and from 12.5 pg/10(6) cells to 1235.4 pg/10(6) cells for primary leukemic cells from ALL patients (N=13). The presence of histamine in the cytoplasm of these ALL cells was also confirmed by immunostaining using a polyclonal rabbit anti-histamine antibody. Notably, the histamine receptor blocker diphenhydramine inhibited the clonogenic growth of ALL cells by >90% prompting the hypothesis that histamine may be an autocrine regulator of ALL cell proliferation. Our study suggests that histamine receptor blockers may therefore be useful for the treatment of therapy-refractory ALL.

Role of circulating immune complexes in the immunopathogenesis of silicosis.

The sera of 19 silica-dust-exposed subjects and of an equal number of age-, sex- and socioeconomic-strata-matched controls were analysed for antinuclear factor, rheumatoid factor, C-reactive protein, immunoglobulins G, M, A, and complement C3 and C4. Circulating immune complexes were also precipitated in all subjects and their immunoglobulin and complement C3 and C4 were estimated. Silica-exposed subjects were divided into two groups depending upon the radiological findings and it is suggested that IgA plays an important role in the immunopathogenesis of the disease and that lung changes could be due to the immune-complex-mediated mechanisms utilizing an alternative complement pathway.

Effect of high dose intravenous pulse dexamethasone on lymphocyte phenotypes.

The lymphocyte phenotypes were enumerated in 10 patients with collagen diseases at 0 h, 4 h, 24 h and 7 days after a megadose (100 mg) iv pulse dexamethasone. A significant decrease in CD3 (from a mean of 2324.3/mm3 to 705.9/mm3) and CD4 (from a mean of 1642.6 to 317.6/mm3) cells was observed at 4 h, which recovered partially by 24 h (186.7 and 1226.3/mm3 respectively) and completely at 7 days (2496.1 and 1838.4/mm3). A transient decrease in CD8 cells at 4 h was also observed. There was no significant effect on B cells.

Intermittent intravenous pulse cyclophosphamide treatment in systemic lupus erythematosus.

To determine the efficacy and safety of intermittent intravenous pulse cyclophosphamide in patients of severe systemic lupus erythematosus (SLE), 50 patients having severe/refractory lupus nephritis, vasculitis or neuropsychiatric manifestations were treated with 3 weekly pulses of cyclophosphamide for 6 such pulses. This treatment was found to be associated with significant and sustained improvement during a 2 yr follow up with respect to the mean renal activity score, individual renal parameters (proteinuria, erythrocyturia, and serum creatinine levels), focal neurological manifestations, vasculitic lesions, antinuclear antibody titers, complement component C3, anti-dsDNA antibodies levels and ESR. There was a sustained decrease in the overall mean disease activity score, and the mean daily dose of prednisolone (pretreatment 32.62 mg daily to 3.75 mg daily after 24 months). There was a significant decline in the percentage and absolute B cell count after 7, 14 and 21 days of this treatment. Effect on other lymphocyte subsets (CD3+, CD4+ and CD8+) was not marked. Pulse cyclophosphamide could therefore be an effective and less toxic form of treatment in patients with SLE having severe lupus nephritis, focal neurological lesions or vasculitis.

Methotrexate: clinical and immunological effects in refractory rheumatoid arthritis.

Thirty five patients with refractory rheumatoid arthritis were given 7.5 mg of methotrexate (Mtx) every week. Eleven patients had to discontinue treatment either because of adverse effects or unresponsiveness. Twenty four patients showed clinical response and significant improvement in ESR and they continued Mtx for a mean of 25.24 months. Seven patients achieved clinical remission as defined by ARA criteria. Immunological parameeters including IgG, IgM, IgA, lymphocyte subsets (CD3+, CD4+, CD8+ and B), C3 and C4 however, did not show any change during this treatment in any of the groups upto 6 months. There was a significant fall in the erythrocyte sedimentation rate (ESR), c-reactive protein (CRP) and rheumatoid factor (RF) levels in responders only.

AIDS screening in North India: clinical spectrum of HIV infection.

PIP: 8 subjects with human immunodeficiency virus (HIV) infection were encountered between June-December 1986 and were diagnosed as having AIDS (3), PGL (1), MLA (1), and asymptomatic HIV carrier state (3). The clinical presentation, immunologic features, and course of those with AIDS or PGL, were similar to those reported from American, European, and tropical African countries, with low T-helper cells, reversed CD4/CD8 ratio, and the presence of antibody to HIV. Asymptomatic carriers also had reversed CD4/CD8 ratio. 6 of these individuals were foreign visitors, 5 from tropical African countries and 1 from the US, while 2 were Indians who had frequent sexual exposure abroad in countries where AIDS is quite prevalent (1 homosexual in West Germany and other possibly had sexual exposure in Uganda). None of the 2046 Indian nationals in the high risk group screened until January 1987 without history of sexual, blood, or blood product exposure abroad were found to seropositive at this center in North India. These findings suggest that HIV infection is not endemic in North India. However, there is a risk of spread of this infection in North India through sexual or blood contact with foreign visitors.^ieng

Effect of D-penicillamine on lymphocyte subsets: correlation with clinical response in rheumatoid arthritis.

We studied the effects of D-penicillamine (DP) on the clinical response, immunoinflammatory parameters and the lymphocyte subsets in 46 patients with rheumatoid arthritis (RA). Patients were evaluated before the start of the drug and then at 3 and 9 months during the follow up. 38 of 46 (82.6%) patients could continue DP treatment for over 9 months, while in 8 the drug was withdrawn due to adverse effects. Improvement in the various disease activity indices of more than 50% (responders) was seen in 25 of 38 (65.8%) patients. Responders showed a significant decrease in the serum IgA and IgM at 9 months, and in IgM only at 3 months. The serum levels of C3 and C4 did not show any significant change. Serum levels of C-reactive protein and rheumatoid factor (RF) showed a significant decrease at 3 and 9 months. A significant decrease in CD3+ and CD4+ lymphocytes along with a fall in CD4+/CD8+ lymphocyte ratio was also seen in responders at 3 and 9 months, compared to the baseline. Our results suggest that DP may have immunomodulatory action in RA.

Anti-inflammatory activity of 2,4, 6-trihydroxy-alpha-p-methoxyphenyl-acetophenone (compound D-58).

BACKGROUND: Active oxygen radicals as well as a variety of cytosolic protein tyrosine kinases play a role in the regulation of prostaglandin E(2) (PGE(2)), a key inflammatory mediator, released by skin cells in response to irradiation with ultraviolet B light (UVB). Identification of chemical compounds that can interrupt such events may provide a basis for the development of potent anti-inflammatory agents. OBJECTIVE: To investigate the effect of a novel genistein analog, 2,4, 6-trihydroxy-alpha-p-methoxyphenylacetophenone, with antioxidant property (compound D-58), on UVB-induced inflammatory responses. METHODS: Epidermal cell cultures were irradiated with UVB both in the presence and absence of compound D-58 and the PGE(2) released in the medium was determined by ELISA. For in vivo studies, skin inflammation was induced in mice either by carrageenan challenge of the air pouch or by an acute exposure of skin to UVB radiation. The resulting inflammatory mediator release, skin edema and the histological changes of the skin were determined both in the presence and absence of compound D-58. RESULTS: Compound D-58 treatment effectively inhibited the development of edema and histological changes in the skin of UVB-irradiated mice as well as the release of PGE(2) in vitro as well as in vivo. CONCLUSION: Compound D-58 (2,4,6-trihydroxy-alpha-p-methoxyphenylacetophenone) has potent anti-inflammatory properties.

Reversible translocation of 5-lipoxygenase in mast cells upon IgE/antigen stimulation.

The 5-lipoxygenase catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes. It has been shown that the calcium influx into leukocytes following stimulation by A23187 activates the enzyme and causes its translocation to the membrane. Leukotrienes are formed, and then the enzyme seems to be irreversibly inactivated. In the present investigation we have compared the effect of receptor-mediated mast cell activation (IgE/antigen) and A23187 on 5-lipoxygenase activity and translocation to the membrane. In contrast to the ionophore, IgE/antigen, which initiated the formation of similar amounts of leukotrienes as A23187, caused minor inactivation of the enzyme. Immunoblot analysis demonstrated that after antigen the membrane association of the 5-lipoxygenase was reversible, while with A23187 the translocation continued during the time of observation (60 min). Addition of a calcium chelator after ionophore challenge, prevented further inactivation of the enzyme and reversed its membrane binding. The data suggest that the continuous influx of calcium with A23187 is responsible for the extensive inactivation of the 5-lipoxygenase. In contrast, during receptor-mediated stimulation the transient increase in intracellular calcium seems to conserve the enzyme activity.

Control of mast-cell 5-lipoxygenase.

Leukotrienes (LTs) from mast cells make important contributions to early events in inflammation. Therefore, the control of their 5-lipoxygenase was studied. We observed that culture conditions can significantly alter LT synthesis and intracellular 5-lipoxygenase levels. Challenge of mast cells with calcium ionophore A23187 and immunoglobin E (IgE)/antigen had different effects. The calcium ionophore caused continuous LT formation that was accompanied by the translocation of the enzyme to membranes as well as substantial loss of activity. In contrast, with receptor-mediated stimulation, IgE/antigen, enzyme inactivation was insignificant, and membrane binding and LT synthesis were transient. Addition of a calcium chelator stopped ionophore-induced LT production and inactivation of the 5-lipoxygenase and reversed its membrane association. Therefore, receptor-mediated activation of 5-lipoxygenase differs from that by calcium ionophore. The data also indicate that calcium regulates the membrane binding and dissociation of the 5-lipoxygenase. However, with excessively high calcium concentrations activity is lost.