Allergic eosinophil-rich inflammation develops in lungs and airways of B cell-deficient mice.

Immunoglobulins (Ig), particularly IgE, are believed to be crucially involved in the pathogenesis of asthma and, equally, in allergic models of the disease. To validate this paradigm we examined homozygous mutant C57BL/6 mice, which are B cell deficient, lacking all Ig. Mice were immunized intraperitoneally with 10 micrograms ovalbumin (OVA) plus alum, followed by daily (day 14-20) 30 min exposures to OVA aerosol (OVA/OVA group). Three control groups were run: OVA intraperitoneally plus saline (SAL) aerosol (OVA/SAL group); saline intraperitoneally plus saline aerosol; saline intraperitoneally plus OVA aerosol (n = 6-7). Lung and large airway tissues obtained 24 h after the last OVA or SAL exposure were examined by light microscopy and transmission electron microscopy (TEM). The Ig-deficient mice receiving OVA/ OVA treatment had swollen and discolored lungs and exhibited marked eosinophilia both in large airway subepithelial tissue (49.2 +/- 12.0 cells/mm basement membrane [BM] versus OVA/ SAL control 1.2 +/- 0.3 cells/mm BM; P < 0.001), and perivascularly and peribronchially in the lung (49.3 +/- 9.0 cells/unit area versus OVA/SAL control 2.6 +/- 0.6 cells/unit area; P < 0.001). The eosinophilia extended to the regional lymph nodes. TEM confirmed the subepithelial and perivascular localization of eosinophils. Mucus cells in large airway epithelium increased from 1.5 +/- 0.8 (OVA/SAL mice) to 39.5 +/- 5.7 cells/mm BM in OVA/OVA treated mice (P < 0.001). OVA/SAL mice never differed from the other control groups. Corresponding experiments in wild-type mice (n = 6-7 in each group) showed qualitatively similar but less pronounced eosinophil and mucus cell changes. Macrophages and CD4+ T cells increased in lungs of all OVA/OVA-treated mice. Mast cell number did not differ but degranulation was detected only in OVA/OVA-treated wild-type mice. Immunization to OVA followed by OVA challenges thus cause eosinophil-rich inflammation in airways and lungs of mice without involvement of B cells and Ig.

Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice.

The earliest contact between antigen and the innate immune system is thought to direct the subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune system, such as natural killer (NK) cells, NK1.1(+) T cells (NKT cells), and gamma/delta T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of NK1.1(+) cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil and CD3(+) T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon gamma-producing splenic cells were diminished in mice depleted of NK1.1(+) cells before the priming regime. Depletion of NK1.1(+) cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different from those observed in wild-type mice. Mice deficient in gamma/delta T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a pathogenic role for the increased NK cell activity observed in human asthma.

A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis.

AIMS/HYPOTHESIS: The enzyme hormone-sensitive lipase (HSL) is produced and is active in pancreatic beta cells. Because lipids are known to play a crucial role in normal control of insulin release and in the deterioration of beta cell function, as observed in type 2 diabetes, actions of HSL in beta cells may be critical. This notion has been addressed in different lines of HSL knockout mice with contradictory results. METHODS: To resolve this, we created a transgenic mouse lacking HSL specifically in beta cells, and characterised this model with regard to glucose metabolism and insulin secretion, using both in vivo and in vitro methods. RESULTS: We found that fasting basal plasma glucose levels were significantly elevated in mice lacking HSL in beta cells. An IVGTT at 12 weeks revealed a blunting of the initial insulin response to glucose with delayed elimination of the sugar. Additionally, arginine-stimulated insulin secretion was markedly diminished in vivo. Investigation of the exocytotic response in single HSL-deficient beta cells showed an impaired response to depolarisation of the plasma membrane. Beta cell mass and islet insulin content were increased, suggesting a compensatory mechanism, by which beta cells lacking HSL strive to maintain normoglycaemia. CONCLUSIONS/INTERPRETATION: Based on these results, we suggest that HSL, which is located in close proximity of the secretory granules, may serve as provider of a lipid-derived signal essential for normal insulin secretion.

A substance P antagonist, [D-Pro2, D-Trp7,9]SP, inhibits inflammatory responses in the rabbit eye.

Neurogenic factors released by antidromic nerve stimulation are thought to be in part responsible for the vasodilation and breakdown of the blood-aqueous barrier that follows trauma to the eye. Substance P is one candidate for the mediation of the inflammatory response since it is thought to be a neurotransmitter in sensory afferents and since exogenous substance P is capable of eliciting a response characteristic of inflammation. In rabbits, intravitreal or topical application onto the eye of a specific substance P antagonist, [d-Pro2, D-Trp7,9]SP, inhibited not only the irritant effects of exogenous substance P but also the inflammatory response to a standardized trauma (infrared irradiation of the iris). These observations suggest that substance P, or a related peptide, is a neurogenic mediator of the inflammatory response in the eye.

Reconstructing the pancreas: restoration of normoglycemia, exocrine function, and islet innervation by islet transplantation to the pancreas.

Impaired function in transplanted islets may be ascribed in part to disturbed reinnervation. The objectives of this study were to determine whether islet transplantation to the pancreas in the presence of nerve growth factor (NGF) would restore islet innervation and endocrine and exocrine pancreatic function. Streptozotocin-diabetic Lewis rats received 800 syngeneic islets beneath the pancreatic capsule in the presence or absence of NGF (20 ng/d for 14 days). Fasting blood glucose was measured for 3 months. The pancreata were isolated and perfused in situ. Pancreatic juice was collected for amylase determination. The sympathetic trunks were isolated and stimulated electrically. The tissues were immunostained for nerve markers. All islet recipients remained euglycemic (4.2 +/- 0.6 mmol/L glucose). Ductal amylase concentrations were restored to near normal levels in contrast to diabetic controls (normal rat 98 +/- 8 U/L, islet transplant 78.4 +/- 9 U/L, diabetic control 14.5 +/- 8 U/L). NGF enhanced the innervation of transplanted islets in contrast to control islet transplants. Sympathetic adrenergic innervation was significantly increased by NGF (tyrosine hydroxylase [P < .001] and neuropeptide Y [P < .05]). No differences in parasympathetic innervation were observed (vesicular acetylcholine transporter). Electrical stimulation of the sympathetic trunks in the presence of 4 micromol/L phentolamine and 5 micromol/L atropine resulted in increased insulin secretion in NGF-treated islet transplants (164%) compared with control transplants (30%). The combination of growth factors and the pancreatic site may allow the use of fewer islets than conventional islet transplant sites and promote more normal transplanted islet function by the enhancement of islet reinnervation.

Ghrelin and insulin gene expression changes in streptozotocin-induced diabetic rats after rosiglitazone pretreatment.

The aim of the study was to evaluate the effect of rosiglitazone treatment on islet ghrelin and insulin gene expressions in streptozotocin (STZ)-induced diabetic rats. Animals were divided into four groups. 1. Intact controls. 2. Rosiglitazone-treated controls. 3. STZ-induced diabetes. 4. Rosiglitazone-treated diabetes. Rosiglitazone was given for 7 days at a dose of 20 mg/kg body weight. Ghrelin and insulin gene expressions were investigated by immunohistochemistry and in situ hybridization. There was no statistically significant difference in body weight between STZ-induced diabetic rats and rosiglitazone-treated diabetic rats during the experimental period. Furthermore, there were no significant differences in blood glucose levels and insulin immunoreactive cell numbers between STZ-induced diabetic rats and rosiglitazone-treated diabetic rats. There was a tendency towards a reduction of ghrelin gene expression in diabetic animals compared with intact controls. We found, in addition, that ghrelin immunoreactive and ghrelin mRNA expressing cells were frequent in the epithelial lining of the ducts suggesting ductal epithelium might be the source of the regenerating islet ghrelin cells, as is known for other islet cells. The results show that short-term rosiglitazone pretreatment had no significant effect on ghrelin and insulin gene expressions.

Procolipase is produced in the rat stomach--a novel source of enterostatin.

Procolipase was identified in the stomach by in situ hybridisation. A strong autoradiographic labelling of chief cells was seen in the fundus region, declining more distally and being almost absent in antrum. There was no labelling seen in the intestine. Colipase activity was estimated in rat gastric juice following pentagastrin stimulation and was found to average 2 microM. Furthermore, enterostatin, the N-terminal pentapeptide of procolipase, has been identified in the rat gut and pancreas. Extracts from gastric mucosa, intestinal mucosa and pancreas were purified by gel filtration (Sephadex G25), ion-exchange chromatography (CM-Sepharose) and HPLC (C18 reverse phase). Using an ELISA assay with antibodies directed against enterostatin, two forms of the peptide were identified both in the gut and in the pancreas, with the amino-acid sequences APGPR and VPGPR, respectively. APGPR was found to be the predominant form of enterostatin, whereas only a small amount had the structure VPGPR. Enterostatin in the form of APGPR, when injected intracerebroventricularly in female Sprague-Dawley rats, significantly reduced high-fat food intake in a two-choice situation of low-fat (14% fat by energy) and high-fat (38% fat) food. It is concluded that procolipase is produced in the stomach and secreted into the gastric juice. This is also a novel source of enterostatin.

The mouse trap.

Mouse models of asthma are now being used extensively in drug research. However, the successful unravelling of combinatorial interplays of cells and molecules in the murine airways may not be matched by equally successful demonstrations of an asthma-like pathophysiology. Here, Carl Persson, Jonas Erjefält, Magnus Korsgren and Frank Sundler discuss the fact that major features of asthma may still need to be demonstrated in the airways of allergic mice.

Dexamethasone-induced neuropeptide Y expression in rat islet endocrine cells. Rapid reversibility and partial prevention by insulin.

Neuropeptide Y (NPY) is a widely distributed neurotransmitter in the central and peripheral nervous system. In the normal rat pancreas, NPY is confined to neuronal elements, including fibers penetrating the islets. However, treatment of rats with the glucocorticoid dexamethasone (DEX) induces NPY expression also in islet cells. Previously performed double immunocytochemistry (ICC) and in situ hybridization (ISH) combined with ICC revealed that the majority of NPY-expressing islet cells are beta-cells. The present study, using ICC, ISH, and Northern blot, addressed the question whether the islet cell expression of NPY induced by DEX is affected by concomitant insulin (60 U/kg body wt daily for 12 days) treatment. Further, the time course of NPY expression in the islet cells after DEX withdrawal was examined. Treatment with DEX (2 mg/kg body wt daily for 12 days) confirmed the induction of NPY expression in numerous cells, most of which were beta-cells, dispersed within the islets. Northern blot analysis of RNA extracted from isolated islets of DEX-treated rats revealed a strong signal for NPY. Furthermore, DEX also induced NPY expression in isolated rat islets during a 5-day culture period in DEX (100 nmol/l). In vivo, the DEX-induced islet cell expression of NPY mRNA was rapidly reversed after cessation of DEX, being nondetectable 5 days post-treatment; NPY peptide was nondetectable 10 days post-treatment, indicating a slower turnover of the formed peptide. After combined treatment with DEX and insulin, the frequency of islet cells expressing NPY was markedly lower than after treatment with DEX alone. The vast majority of the NPY-expressing cells were beta-cells. In conclusion, DEX-induced NPY expression in rat islet cells is dependent on continuous DEX treatment and is partly prevented by exogenous insulin. The results suggest that the DEX-induced islet NPY expression is regulated by insulin.

Reinnervation of syngeneic mouse pancreatic islets transplanted into renal subcapsular space.

A syngeneic transplantation of 150 islets into the subcapsular renal space was performed on normoglycemic or alloxan-induced diabetic male C57BL/6 mice. Six, 8, 14, or 20-21 wk after transplantation, the graft-bearing kidney was removed and processed for microscopical examinations with indirect immunofluorescence for neuropeptides and tyrosine hydroxylase, and with acetylcholinesterase staining to visualize nerve fibers within the graft. Six weeks after implantation, only a few scattered nerve fibers were observed within the grafts. A progressive increase in the number of nerves was observed until 14 wk after transplantation, after which, a stable level was reached. Alloxan-induced diabetic mice showed quantitatively and qualitatively similar reinnervation to normoglycemic mice 20 wk after transplantation. The findings demonstrate the presence of sympathetic nerve fibers (containing tyrosine hydroxylase and neuropeptide Y), mainly accompanying ingrowing blood vessels; parasympathetic nerve fibers (containing acetylcholinesterase and vasoactive intestinal peptide), possibly reaching the graft from the adjacent renal capsule; and afferent nerve fibers (containing substance P and calcitonin gene-related peptide), which were less numerous. The data suggest that transplanted islets become reinnervated by ingrowth of nerve fibers from the implantation organ and that several types of nerves are present.