Cytokine Concentrations In Bronchoalveolar Lavage Fluid Research Articles

Methods for Experimental Allergen Immunotherapy: Subcutaneous and Sublingual Desensitization in Mouse Models of Allergic Asthma.

Allergic asthma is characterized by airway hyperresponsiveness, remodeling, and reversible airway obstruction. This is associated with an eosinophilic inflammation of the airways, caused by inhaled allergens such as house dust mite or grass pollen. The inhaled allergens trigger a type-2 inflammatory response with the involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high immunoglobulin E (IgE) antibody production by B cells and mucus production by airway epithelial cells. As a consequence of the IgE production, subsequent allergen reexposure results in a classic allergic response with distinct early and late phases, both resulting in bronchoconstriction and shortness of breath. Allergen-specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma. Both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have shown clinical efficacy in long-term suppression of the allergic response. Although AIT treatments are very successful for rhinitis, application in asthma is hampered by variable efficacy, long duration of treatment, and risk of severe side effects. A more profound understanding of the mechanisms by which AIT induces tolerance to allergens in sensitized individuals is needed to be able to improve its efficacy. Mouse models have been very valuable in preclinical research for characterizing the mechanisms of desensitization in AIT and evaluating novel approaches to improve its efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen adsorbed to aluminum hydroxide, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of allergen extracts as an immunotherapy treatment. Finally, mice are challenged by intranasal allergen administrations. We will also describe the protocols as well as the most important readout parameters for the measurements of invasive lung function, serum immunoglobulin levels, isolation of bronchoalveolar lavage fluid (BALF), and preparation of cytospin slides. Moreover, we describe how to perform ex vivo restimulation of lung single-cell suspensions with allergens, flow cytometry for identification of relevant immune cell populations, and ELISAs and Luminex assays for assessment of the cytokine concentrations in BALF and lung tissue.

Protective effect of ulinastatin on severe pulmonary infection under immunosuppression and its molecular mechanism.

The objective of the present study was to investigate the protective effect of ulinastatin on severe pulmonary infection under immunosuppression, and its molecular mechanism. Mice were treated with methylprednisolone and lipopolysaccharide (LPS) to establish the model of severe pulmonary infection under immunosuppression. Mice were randomly divided into group A (model group; treated with equal volumes of saline), group B (treated with 1×105 U/kg ulinastatin), and group C (normal control group). Bronchoalveolar lavage fluid (BALF) was collected, and the concentrations of cytokines in BALF were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in lung tissues were observed by hematoxylin and eosin (H&E) staining. The mRNA levels of M1 and M2 macrophage markers in lung tissues were detected by real-time polymerase chain reaction (PCR). Specific protein levels in lung tissues were measured by western blotting. Apoptosis in lung tissues was detected by the terminal-deoxynucleoitidyl transferase mediated nick end-labeling (TUNEL) method. The concentrations of TNF-α, IL-6, and IL-1β in BALF, the mRNA levels of the three M1 macrophage markers, and the protein levels of p-Janus Kinase 2 (p-JAK2), p-signal transducer and activator of transcription-3 (p-STAT-3), cleaved caspase-9, and cleaved poly-ADP-ribose polymerase (PARP), and the number of apoptotic cells in lung tissues in group A were significantly higher than those in groups B and C (P<0.05), whereas the concentrations of IL-4, IL-10, and IL-13 and the mRNA levels of the three M2 macrophage markers were significantly lower than those in groups B and C (P<0.05). Immunofluorescence showed that the nuclei of lung epithelial macrophages in group A became smaller and moved towards the side of nuclear membranes. In conclusion, ulinastatin can improve the inflammatory response caused by severe infection under immunosuppression, which balances the inflammatory microenvironment and inhibits apoptosis at least partially through inhibiting JAK2/STAT-3 and/or caspase pathway activity, ultimately playing a role in lung protection.

Subcutaneous and Sublingual Immunotherapy in a Mouse Model of Allergic Asthma.

Allergic asthma, caused by inhaled allergens such as house dust mite or grass pollen, is characterized by reversible airway obstruction, associated with an eosinophilic inflammation of the airways, as well as airway hyper responsiveness and remodeling. The inhaled allergens trigger a type-2 inflammatory response with involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high production of immunoglobulin E (IgE) antibodies. Consequently, renewed allergen exposure results in a classic allergic response with a distinct early and late phase, both resulting in bronchoconstriction and shortness of breath. Allergen specific immunotherapy (AIT) is the only treatment that is capable of modifying the immunological process underlying allergic responses including allergic asthma and both subcutaneous AIT (SCIT) as well as sublingual AIT (SLIT) have proven clinical efficacy in long term suppression of the allergic response. Although these treatments are very successful for rhinitis, application of AIT in asthma is hampered by variable efficacy, long duration of treatment, and the risk of severe side-effects. A more profound understanding of the mechanisms by which AIT achieves tolerance to allergens in sensitized individuals is needed to improve its efficacy. Mouse models have been very valuable as a preclinical model to characterize the mechanisms of desensitization in AIT and to evaluate novel approaches for improved efficacy. Here, we present a rapid and reproducible mouse model for allergen-specific immunotherapy. In this model, mice are sensitized with two injections of allergen absorbed to aluminum hydroxide to induce allergic sensitization, followed by subcutaneous injections (SCIT) or sublingual administrations (SLIT) of the allergen as immunotherapy treatment. Finally, mice are challenged by three intranasal allergen administrations. We will describe the protocols as well as the most important read-out parameters including measurement of invasive lung function measurements, serum immunoglobulin levels, isolation of broncho-alveolar lavage fluid (BALF), and preparation of cytospins. Moreover, we describe how to restimulate lung single cell suspensions, perform flow cytometry measurements to identify populations of relevant immune cells, and perform ELISAs and Luminex assays to measure the cytokine concentrations in BALF and lung tissue.

Cytokine concentrations in bronchoalveolar lavage fluid from horses with neutrophilic inflammatory airway disease.

BackgroundMultiple cytological patterns occur in bronchoalveolar lavage fluid (BALF) of horses with inflammatory airway disease (IAD). Only few data on BALF cytokine profiles are available for horses with IAD, and are limited to mRNA expression.Hypothesis/ObjectiveCytological profiles of IAD are associated with different BALF immunological pathways. To investigate BALF cytokine concentrations in a large number of horses with neutrophilic IAD.AnimalsOne hundred and thirty‐eight client‐owned Standardbred racehorses in active training.MethodsProspective observational study. BALF samples were obtained from left and right lungs. Interleukin (IL)‐4, interferon (IFN)‐γ, and tumor necrosis factor (TNF)‐α concentrations were determined by ELISA.ResultsFourteen horses had normal BALF cytological profiles and 56 exhibited evidence of bilateral neutrophilic IAD. Twenty‐four horses showed BALF with, respectively, IAD‐ and CTL consistent cytology and were excluded; as were 44 horses because of evidence of pulmonary hemorrhage. TNF‐α (56 ± 115 pg/mL; P = .034) and IFN‐γ concentrations (104 ± 247 pg/mL; P = .044) were significantly higher for IAD horses, compared with controls (respectively 19 ± 41 and 80 ± 116 pg/mL). Horses with ‘neutrophil’ subtype had significantly higher IFN‐γ concentrations (110 ± 154 pg/mL), than ‘neutrophil/metachromatic’ (56 ± 54 pg/mL; P = .028) and ‘neutrophil/metachromatic/eosinophil’ subtypes (44 ± 23 pg/mL; P = .012).Conclusions and Clinical ImportanceCytokine concentrations in BALF suggested that neutrophilic IAD is associated with activation of the innate immune system and a possible T‐helper (Th)‐1 polarized response. This study also suggested that immunological pathways vary according to cytological IAD subtypes.

Dok adaptors play anti‐inflammatory roles in pulmonary homeostasis

Asthma is a chronic inflammatory disease of the lung with airflow obstruction and bronchospasm, characterized by pulmonary eosinophilia, airway remodeling, increased airway hyperresponsiveness to environmental stimuli, and excessive Th2-type cytokine production. Recent studies indicate that crosstalk between the innate and adaptive immune systems is crucial for this disease. We and others have showed that the Dok (downstream of tyrosine kinases) family adaptors, Dok-1, Dok-2, and Dok-3, play essential roles in negative regulation of a wide variety of signaling pathways in both innate and adaptive immunities. Here, histopathology and bronchoalveolar lavage fluid (BALF) cellularity showed spontaneous pulmonary inflammation in Dok-1-/- Dok-2-/- Dok-3-/- (TKO) mice, but not in Dok-1-/- Dok-2-/- or Dok-3-/- mice, with hallmarks of asthma, including eosinophilia, goblet cell hyperplasia, and subepithelial fibrosis. Consistently, TKO mice, but not the other mutants, showed increased airway hyperresponsiveness to methacholine inhalation. In addition, Th2-type cytokine concentrations in BALF were increased in TKO mice. These findings provide strong evidence that Dok-1, Dok-2, and Dok-3 cooperatively play critical anti-inflammatory roles in lung homeostasis.

Adjuvanted rush immunotherapy using CpG oligodeoxynucleotides in experimental feline allergic asthma

Allergic asthma is driven by relative overexpression of Th2 cell-derived cytokines in response to aeroallergens. In independent studies, both allergen-specific rush immunotherapy (RIT) and CpG oligodeoxynucleotides (ODN) showed promise in blunting eosinophilic inflammation in a model of feline allergic asthma. We hypothesized that RIT using allergen and CpG ODN would work synergistically to dampen the asthmatic phenotype in experimentally asthmatic cats. Twelve cats with asthma induced using Bermuda grass allergen (BGA) were studied. Of these, six were administered adjuvanted BGA RIT using CpG ODN #2142; six were administered placebo (saline) RIT and later crossed over to adjuvanted RIT. Over 2 days, subcutaneous CpG ODN (0.5 ng/kg) with BGA (increasing doses every 2 h from 20 to 200 μg) was administered. Adverse events were recorded and compared with historical controls. Percentage of eosinophils in bronchoalveolar lavage fluid (BALF), % peripheral CD4 + CD25+ T regulatory cells (Tregs), lymphocyte proliferation in response to ConA, and cytokine concentrations in BALF were measured over 2 months. Group mean BALF % eosinophils for the adjuvanted RIT cats were significantly lower at week 1 and month 1 ( p = 0.03 for both), and marginally significantly lower at month 2 ( p = 0.09) compared with placebo RIT cats. By the end of the study, 8/12 treated cats had BALF % eosinophils within the reference range for healthy cats. Adjuvanted RIT, but not placebo RIT, cats had significant decreases in the ConA stimulation index over time ( p = 0.05). BALF IL-4 concentrations were significantly higher at week 1 in adjuvanted RIT cats compared with baseline and month 2, and also with placebo RIT cats at week 1. No significant differences were detected between treatments or over time for IL-10 or IFN-γ concentrations in BALF or for %Tregs cells in peripheral blood. Adjuvanted RIT using CpG ODN in experimental feline asthma dampens eosinophilic airway inflammation. Adverse effects associated with adjuvanted RIT were less severe compared with a historical, non-adjuvanted RIT protocol. The exact mechanism(s) by which adjuvanted RIT alters the aberrant allergic immune response were not elucidated in this study.

Dose and Time Dependency of Inflammatory Responses in the Mouse Lung to Urban Air Coarse, Fine, and Ultrafine Particles From Six European Cities

We investigated the dose and time dependency of inflammatory and cytotoxic responses to size-segregated urban air particulate samples in the mouse lung. Coarse (PM10−2.5), fine (PM2.5−0.2), and ultrafine (PM0.2) particles were collected in six European cities (Duisburg, Prague, Amsterdam, Helsinki, Barcelona, Athens) in selected seasons using a modified Harvard high-volume cascade impactor. Healthy C57Bl/6J mice were intratracheally exposed to the particulate samples in a 24-h dose-response study (1, 3, and 10 mg/kg) and in 4-, 12-, and 24-h time course studies (10 mg/kg). After the exposures, the lungs were lavaged and the bronchoalveolar lavage fluid (BALF) was assayed for indicators of inflammation and tissue damage: total cell number, cell differential, total protein, and lactate dehydrogenase (LDH) and cytokine (tumor necrosis alpha [TNF-α], interleukin-6 [IL-6], and keratinocyte-derived chemokine [KC]) concentrations. In general, PM10−2.5 samples had higher inflammatory activity than PM2.5−0.2 samples. PM0.2 samples showed negligible inflammatory activity. PM10−2.5 and PM2.5−0.2 samples caused large increases in BALF cytokine concentrations at 4 h, but not at 12 or 24 h, after exposure. The BALF total cell number and total protein concentrations increased significantly at 12 h for both the PM10−2.5 and PM2.5−0.2 samples, but only PM10−2.5 samples produced consistent, significant increases at 24 h after exposure. There was more heterogeneity in BALF cytokine and neutrophil cell number responses to PM2.5−0.2 samples than to PM10−2.5 samples between the sampling campaigns. Thus, particle size, sources, and atmospheric transformation processes affect the inflammatory activity and response duration of urban air particulate matter in the mouse lung.

Alterations of mononuclear inflammatory cells, CD4/CD8+ T cells, interleukin 1β, and tumour necrosis factor α in the bronchoalveolar lavage fluid, peripheral blood, and skin of patients with systemic sclerosis

Background: Systemic sclerosis (SSc) is a multisystem disease with underlying immune mechanisms. Aims: To investigate the clinicopathological characteristics of the lesions; immunological alterations in the bronchoalveolar lavage fluid (BALF), peripheral...