Primary sensitization to inhalant allergens.

The neonatal T-cell system is capable of responding to allergens at birth, indicating the occurrence of prenatal sensitization, and the cytokine profile of these responses is skewed towards the Th-2 type. This response is further modified by postnatal exposure to different types of allergens. In relation to inhalant allergen (employed by HDM) the low level fetal Th-2 responses in non-atopics appear to be down-regulated rapidly after birth, parallel to an increase in allergen-specific IFN-gamma production. In contrast, atopics appear to consolidate their initial Th-2 responses, and around the age of 6 exhibit a cytokine response profile similar to the adult pattern. A pre-existing deficiency in IFN-gamma production may be one of the key factors determining the postnatal persistence of Th-2 responses in atopics.

Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity.

Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the "default" T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2-3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-gamma by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing- associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor alpha or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.

Epithelial cell damage is induced by neutrophil-derived, not pseudomonas-derived, proteases in cystic fibrosis sputum.

Airway histopathological changes in cystic fibrosis (CF) include damage to the epithelial tissue and accumulation of polymorphonuclear leukocytes (PMN). Airways of CF patients are usually colonized with bacteria such as mucoid Pseudomonas aeruginosa (PA). Bacteria and PMN can both release proteolytic enzymes capable of causing tissue damage. This study aims to clarify and compare the roles of these agents in epithelium damage. Epithelial cell (EC) damage and detachment induced by sputum samples from CF or non-CF patients, with and without lung infection, were assessed on amnionic EC in an in vitro model of airway epithelium. Protease activity was determined using inhibitor profiles, and compared to the proteolytic activity of isolated neutrophils and bacteria. Sputa from CF patients and infected non-CF patients induced high levels of detachment. PA also induced high levels of EC detachment but Staphylococcus aureus and Haemophilus influenzae, two other bacteria commonly isolated from CF sputa, induced no detachment. Antiprotease inhibition profiles were similar for PMN and sputa-induced EC detachment, but different for PA-induced detachment. These results suggest that PMN proteolytic enzymes, probably elastase and cathepsin G, are more likely to be the inducers of tissue damage in the airways of CF patients than PA proteolytic enzymes.

Developing patterns of T cell memory to environmental allergens in the first two years of life.

Several recent studies have demonstrated cord blood mononuclear cell (CBMC) proliferation in response to food and inhalant allergens, suggesting that initial T-cell-priming may occur in utero. The findings below from an ongoing prospective study on 60 subjects provide initial information on the nature of accompanying T cell cytokine responses. We demonstrate CBMC proliferation following culture with house dust mite and ovalbumin (OVA) in 47 and 42% of subjects, respectively, compared to an overall rate of 3% for tetanus toxoid; the frequencies of these responses were comparable in neonates with and without atopic family history (FH). With the exception of IL-10, analysis of cytokine responses in allergen-stimulated cultures of CBMCs required the use of semiquantitative RT-PCR, which revealed low-level IL-4 and/or IL-5 mRNA production, in particular a 50% IL-5 response rate to OVA in FH-positive neonates. IFN-gamma responses were less frequent and required higher PCR cycle numbers for detection. Preliminary analysis of culture supernatants from a subgroup of CBMCs indicate high-level allergen-specific IL-10 responses in both FH-negative and -positive subjects, detectable by ELISA. Parallel PCR studies on MCs from 27 children (mean age 18 months) indicated a clear segregation at this age on the basis of FH, with Th0-like or mixed Th1/Th2 responses (IL-5 plus IFN-gamma) which were mainly restricted to the FH-positive group.

TH2-polarized immunological memory to inhalant allergens in atopics is established during infancy and early childhood.

BACKGROUND: There is increasing evidence that the T-cell reactivity to environmental allergens underlying expression of allergic disease in adulthood, develops initially during childhood. However, there is little information available on the kinetics of these early responses, or on the patterns of cytokine production during this period. OBJECTIVE: The purpose of this study was twofold: to obtain further information on the reported differences between responses to food versus inhalant allergens during early childhood, and to ascertain the age-range over which T-cell responses to inhalant allergens become polarized towards the TH2 cytokine profile, in potentially atopic children. METHODS: In vitro cytokine responses to house dust mite (HDM) and egg (OVA) were assessed by semiquantitative RT-PCR in panels of 2- and 5-year-old children and adults; lymphoproliferative responses to OVA were subjected to epitope analysis. RESULTS: At age 2 years IL-4/IL-5 responses to HDM grouped with positive atopic family history, and by age 5 years cytokine responses correlated strongly with individual SPT reactivity to HDM. In contrast, OVA responses were restricted to weak and transient IL-5 signals in the 2-year-old family history positive group. Lymphoproliferation assays performed in parallel indicate a log-scale greater postnatal expansion of T-cell reactivity to the inhalant allergen; preliminary epitope analysis of OVA responses indicate that the number of OVA epitopes recognised decrease during early childhood. CONCLUSIONS: Inhalant allergen-specific in vitro cytokine production associated with positive skin-prick test (SPT) reactions, one of the hallmarks of adult atopy, manifests in children at or before 5 years of age; additionally, cytokine responses in SPT negative 5 year-olds are restricted to IFNgamma, as per normal adults. In contrast, T-cell responses to a typical food allergen appear to be deleted during early childhood.

Role of neutrophils in mediating human epithelial cell detachment from native basement membrane.

Epithelial cell detachment from underlying basement membrane is a feature of diseases of many organs. In the lungs it is seen in disorders as diverse as bronchiectasis, allograft rejection, and asthma. The potential for different leukocytes to induce this change is not clear. In asthma both eosinophils and neutrophils are found in affected tissues, but the capacity of each of these types of cells to induce detachment of native epithelial cells from basement membrane requires clarification. Although eosinophils damage rather than detach human epithelial cells, the effects of neutrophils on epithelial cells naturally attached to basement membrane have not previously been described. Using the human amnion in vitro model, we tested the hypothesis that neutrophils have the capacity to detach intact human epithelial cells from basement membrane. The data indicate that increasing concentrations of neutrophils are able to detach epithelial cells from their underlying basement membrane. Detachment was increased when the neutrophils were activated in situ with tetradecanoyl phorbol acetate and after longer incubation periods. Platelet activating factor and opsonized zymosan showed similar boosting effects, whereas activated complement and formyl-methyl-leucyl-phenylalanine did not. Physical contact of the neutrophils with the epithelial cells was required to induce detachment. Detachment could be inhibited by glutathione and by soybean trypsin inhibitor, an inhibition pattern similar to cathepsin G and trypsin, but not collagenase, in this system. We conclude that neutrophils are capable of detaching human epithelial cells from basement membrane, which in part involves the release of chymotrypsin-like serine proteases, probably in conjunction with oxidants, and that this detachment can be inhibited.

Effects of different density gradient separation techniques on neutrophil function.

Investigation of the pathogenesis of inflammatory diseases has involved assessment of polymorphonuclear leukocyte (PMN) activity and a variety of techniques for separating PMNs from whole blood have been described. In this study the effects of Percoll gradient, Ficoll-Hypaque/Dextran sedimentation (FH/DS) and Mono-Poly Resolving Medium (M-PRM) on activation and function of PMNs were compared. All three separation techniques gave similar cell yield and purity. The mean (+/- SEM) percentage of cells demonstrating pseudopodia formation for Percoll, FH/DS and M-PRM were 39 +/- 9, 57 +/- 6 and 63 +/- 5, respectively, while superoxide release from resting cells was 1.9 +/- 0.9, 7.2 +/- 3.5 and 11 +/- 4.8 pmols per 10(6) cells min-1, respectively, indicating that activation of cells during separation may be less with Percoll compared to the other methods. The functional capacity of the cells to respond to a stimulus was similar for all methods as indicated by similar EC50 values for chemotaxis to zymosan-activated serum and similar superoxide production induced by tetradecanoyl phorbol acetate. All three separation techniques produce functionally active PMNs of high purity but the use of Percoll gradients may be preferable when a quick method of separation which causes minimum pre-activation of PMNs is required.

Allergens as proteases: an Aspergillus fumigatus proteinase directly induces human epithelial cell detachment.

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by pulmonary and systemic allergic and inflammatory processes triggered by fungal antigens. Airway damage is a feature of this disorder, and although Aspergillus-derived proteinases have been described, the capacity of Aspergillus, however, to directly induce damage to human epithelium has not previously been studied. We therefore cultured Aspergillus fumigatus from two patients with ABPA, extracted mycelial products by sonication and filtration, and then evaluated their capacity to induce epithelial cell (EC) desquamation from basement membrane using an in vitro model that uses intact human amniotic EC and native basement membrane. A. fumigatus extracts induced detachment of EC in a dose-dependent fashion, producing up to 34% +/- 6% detachment (p less than 0.05 compared to medium alone). Enzyme analysis of A. Fumigatus extract using synthetic substrates revealed the presence of a number of different enzymes; therefore, studies with specific proteinase inhibitors were undertaken to identify the proteinase(s) responsible for detachment. A. Fumigatus-induced desquamation was partially inhibited by phenylmethylsulfonylfluoride and substantially inhibited by glutathione and N-acetylcysteine, but not by alpha 1-antitrypsin, 1,10 phenanthroline, ethylenediaminetetraacetic acid, aprotinin, or soybean trypsin inhibitor at concentrations that inhibit other serine- or metalloproteinases. Gel filtration of the extract with a Sepharose 6B column revealed that the major epithelium-detaching activity appeared in the 20 to 35 kd fraction. Comparison with proteinase standards suggested a role for a chymotrypsin-like proteinase. Thus, A. fumigatus releases a proteinase that is directly able to induce EC detachment.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of human epithelial cell detachment and damage: effects of proteases and oxidants.

Polymorphonuclear leucocyte (PMN) accumulation is associated with damage to airways epithelial cells in bronchitis, bronchiectasis and some forms of asthma. PMNs release several molecules which may mediate this damage, particularly proteases and oxidants. Using an in vitro model of intact human amnionic epithelial cells (EC) attached to native basement membrane (BM), we evaluated the capacity of several proteases and oxidants to induce detachment of EC from the BM. Maximum desquamation was observed with collagenase, elastase and trypsin, with minimum effective concentrations required to produce 50% EC-desquamation (MEC50) for highly purified collagenase, pancreatic elastase, human leucocyte elastase, human leucocyte cathepsin-G (Cath-G), trypsin, and kallikrein being 3616 +/- 989 U/mL, 32.3 +/- 14.7 U/mL, 85.8 +/- 26.7 U/mL, 360 +/- 20 U/mL, 340 +/- 49 BAEE U/mL and 300 +/- 23 U/mL, respectively. Urokinase (20 U/mL) and plasmin (500 U/mL) produced no desquamation in this system. Relatively high concentrations of oxidants also produced detachment (MEC50 for H2O2 and HOCl being 0.59 +/- 0.006 mol/L and 0.015 +/- 0.009 mol/L, respectively) and pretreatment of EC membranes with non-detaching concentrations of H2O2 rendered them 10-fold more susceptible to protease-induced desquamation, suggesting synergism. Reduced glutathione (GSH), N-acetyl cysteine (NAC), ethylenediamine tetra-acetic acid (EDTA) and 1,10 phenanthroline ablated collagenase induced EC-detachment. Elastase induced detachment was sensitive to inhibition by phenyl methyl sulfonyl fluoride (PMSF) and alpha 1-anti-proteinase (alpha 1-AP) and, to a lesser extent by aprotinin; trypsin-induced detachment was ablated by PMSF, alpha 1-AP and soybean trypsin inhibitor (SBTI) but not by 1,10 phenanthroline or EDTA. Cath-G induced detachment was profoundly inhibited by SBTI, GSH and NAC. These data demonstrate that human EC can be detached from intact BM by several PMN products, including collagenase, Cath-G and elastase, and that PMN-mediated detachment can be prevented by Cath-G and collagenase inhibitors. The data suggest a role for proteases, particularly Cath-G and collagenase, plus oxidants in synergism with proteases, in mediating PMN-induced EC detachment.

Asbestos-induced release of a human alveolar macrophage-derived neutrophil chemotactic factor.

Neutrophils accumulate in the alveoli of asbestos-exposed individuals. In determining whether asbestos fibers induce the release of neutrophil chemotactic factor (NCF) from human alveolar macrophages, alveolar macrophages (10(6) cell/mL) obtained by bronchoalveolar lavage from six non-asbestos-exposed control subjects were exposed to crocidolite (0.1 and 1 mg/mL), chrysotile (1 mg/mL), or medium alone for 4 h, and NCF activity was measured in the supernatants in a 48-well microchemotaxis chamber with polycarbonate membrane filters (pore size, 3 microns) and purified human neutrophils. Alveolar macrophages in medium alone released negligible amounts of NCF (4 +/- 1 neutrophils per high-power field [N/HPF]). When macrophages were exposed to crocidolite (0.1 and 1 mg/mL), significant NCF release occurred (43 +/- 9 and 105 +/- 32 N/HPF, respectively; p less than 0.01 for each amount compare to alveolar macrophages cultured in medium alone). Chrysotile (1 mg/mL) induced similar NCF release (96 +/- 14 N/HPF; p less than 0.01 compared to unstimulated alveolar macrophages). Partial characterization of the NCF by Sephadex G-25 fine gel filtration demonstrated a molecular size of less than 1,000 daltons. These results show that human alveolar macrophages release NCF after exposure to asbestos. Release of NCF by alveolar macrophages in asbestos-exposed individuals may play a central role in the pathogenesis of asbestosis.

Study of human epithelial cell detachment and damage: development of a model.

Detachment of epithelial structures from underlying basement membrane (BM) represents an important component of a number of human disease processes e.g. airway and alveolar diseases, gastrointestinal ulceration, and retinal diseases. This study describes a method of evaluating human epithelial cell detachment from BM that is simple, rapid, inexpensive, quantifiable and which, because it utilizes BM rather than tissue culture plastic, more closely mimics the in vivo situation than other methods. In this model human amnionic epithelial cells attached to their underlying BM are isolated from fresh placentae and mounted in a multi-well chemotaxis assembly. These membranes can be studied with the epithelial cell monolayer intact. Protease-induced detachment of the epithelial cells from the underlying BM was readily quantifiable using light microscopy and spectroscopy. Following removal of the native amnionic epithelial cells, immunoperoxidase staining for the BM attachment proteins laminin, fibronectin, and type IV collagen demonstrated that these molecules remain intact. The BM could also be used as an attachment surface to reconstitute other epithelial cell monolayers. Cultured human amnionic cells and human respiratory epithelial cells were both able to attach to the denuded BM in the absence of serum (% attachment = 85 +/- 15% and 92 +/- 8% respectively, P = 0.8). Natural BM was a better substrate for epithelial cell attachment than tissue culture plastic in that, in the absence of serum, cultured epithelial cell attachment to tissue culture plastic was 20 +/- 4% of the value for BM (P less than 0.05). Furthermore, cells attached to plastic adhered less effectively than to BM in that trypsin concentrations required to induce 50% cell detachment were 0.72 +/- 0.4 for plastic and 62 +/- 13 BAEE U/ml for BM (P less than 0.001). In view of the complex protein interactions known to be involved in the anchorage of human epithelial cells to BM, it is likely this model will be a useful tool for evaluating the mechanisms underlying human epithelial cell attachment and detachment in a variety of normal and disease situations.