Zinc is the toxic factor in the lung response to an atmospheric particulate sample.

The atmospheric dust sample EHC-93 is known to induce lung cell injury and inflammation in which the toxicity has been attributed to a soluble component, possibly metal ions. To determine whether any specific metal is responsible for the pulmonary reactivity, various metal salts, at the concentration of metal present in the soluble fraction of EHC dust, have now been instilled into mouse lung. After 3 days, only a solution containing all metals tested and that of a zinc salt alone induced an increase in inflammatory cells and protein in lung lavage fluid. These two solutions also increased DNA synthesis in lung cells at this time, indicating a reparative response. Other solutions containing metals such as Cu, Fe, Al, Pb, Mg, or Ni induced no changes in the preceding measurements at the EHC dose level of metal. In a more extensive 28-day study, zinc salts induced rapid focal necrosis of Type 1 alveolar epithelial cells followed by inflammation and elevation of protein levels in lavage fluid over a 2-week period. Following the injury, epithelial cell proliferation increased and focal fibrosis was seen at 4 weeks. A solution containing all the other metals tested without the zinc component induced only minimal lung effects. When a zinc salt was administered at a 10x dose, the pulmonary changes were greatly enhanced, and after 4 weeks fibrosis could be measured biochemically. The results indicate that the acute toxicity associated with EHC atmospheric dust is most likely the result of the level of soluble zinc in this particulate sample. This suggests that a high soluble metal content of atmospheric dust, in this case the zinc level, may be a crucial factor in determining pulmonary cell reactivity to inhaled particulates.

Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance.

The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.

Acute pulmonary toxicity of urban particulate matter and ozone.

We have investigated the acute lung toxicity of urban particulate matter in interaction with ozone. Rats were exposed for 4 hours to clean air, ozone (0.8 ppm), the urban dust EHC-93 (5 mg/m3 or 50 mg/m3), or ozone in combination with urban dust. The animals were returned to clean air for 32 hours and then injected (intraperitoneally) with [3H]thymidine to label proliferating cells and killed after 90 minutes. The lungs were fixed by inflation, embedded in glycol methacrylate, and processed for light microscopy autoradiography. Cell labeling was low in bronchioles (0.14 +/- 0.04%) and parenchyma (0.13 +/- 0.02%) of air control animals. Inhalation of EHC-93 alone did not induce cell labeling. Ozone alone increased (P < 0.05) cell labeling (bronchioles, 0.42 +/- 0.16%; parenchyma, 0.57 +/- 0.21%), in line with an acute reparative cell proliferation. The effects of ozone were clearly potentiated by co-exposure with either the low (3.31 +/- 0.31%; 0.99 +/- 0.18%) or the high (4.45 +/- 0.51%; 1.47 +/- 0.18%) concentrations of urban dust (ozone X EHC-93, P < 0.05). Cellular changes were most notable in the epithelia of terminal bronchioles and alveolar ducts and did not distribute to the distal parenchyma. Enhanced DNA synthesis indicates that particulate matter from ambient air can exacerbate epithelial lesions in the lungs. This may extend beyond air pollutant interactions, such as to effects of inhaled particles in the lungs of compromised individuals.

Synthesis, structure-activity relationships, and pharmacokinetic properties of dihydroorotate dehydrogenase inhibitors: 2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'-(trifluoromethyl)phenyl ] propenamide and related compounds.

The active metabolite (2) of the novel immunosuppressive agent leflunomide (1) has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. A series of analogues of the active metabolite 2 have been synthesized. Their in vivo biological activity determined in rat and mouse delayed type hypersensitivity has been found to correlate well with their in vitro DHODH potency. The most promising compound (3) has shown activity in rat and mouse collagen (II)-induced arthritis models (ED50 = 2 and 31 mg/kg, respectively) and has shown a shorter half-life in man when compared with leflunomide. Clinical studies in rheumatoid arthritis are in progress.

Pulmonary response of mice to fiberglass: cytokinetic and biochemical studies.

It has been suggested that glass fibers in the respirable size range may pose a health hazard similar to asbestos because of the similarities in physical characteristics. To compare the pulmonary cell response with that described earlier with crocidolite asbestos, we administered a milled fiberglass sample to mice by intratracheal instillation. Little effect was seen at a dose of 0.1 mg, but at 1 mg there was epithelial injury and an inflammatory cell response concentrated at bronchiolar-alveolar duct regions. Cellular incorporation of tritiated thymidine showed that repair of both bronchiolar and alveolar epithelium occurred rapidly. This was followed by an extended increase in cell labeling, particularly in peribronchiolar fibroblasts, from 2 to 8 wk after fiber instillation. Granulomas formed at this site and later there was morphologic evidence of fibrosis, which was confirmed biochemically by a significant increase in lung collagen at 4-16 wk. Although 10 times higher dose is required, the results show that the lung response to fiberglass in this experimental system is similar to that described previously for crocidolite asbestos; the sites of cell injury and repair are the same, and the subsequent fibrotic response produces small airway disease.

Patterns of particle deposition and retention after instillation to mouse lung during acute injury and fibrotic repair.

Studies on particle deposition, clearance, and translocation to the interstitium and lymph nodes have mostly been carried out on normal animals. This study evaluates changes in these parameters after inert particles are deposited in lungs during acute inflammatory injury and during fibrotic repair. Three days after instilling bleomycin to mouse lungs, there is an inflammatory response and necrosis of type 1 epithelium. When carbon is instilled in these lungs, particles appear uniformly distributed, some are engulfed by the increased phagocytes, but many particles cross the denuded epithelial surface to reach interstitial macrophages. Subsequently much carbon remains in the connective tissue and some reaches hilar lymph nodes. After 16 weeks, particle retention in the lung is significantly greater than in a carbon-only control group. Other mice received carbon 4 weeks after bleomycin when epithelial repair had occurred and many areas of the lung were fibrotic. Very little carbon reached these regions; most was deposited in less fibrotic areas of lung. Few particles crossed the epithelium so that retained carbon in lung and lymph nodes was equivalent to that in the carbon-only group. The results show that a fibrotic lung structure alters the patterns of particle deposition in the lung. However, the major factor determining enhanced particle retention is the integrity of the epithelium. Particle deposition at a time of epithelial injury is associated with enhanced translocation to interstitium and lymph nodes. This may result in pathologic changes if a normally nonreactive low dose of toxic particles is deposited when the epithelium is breached.

The interleukin-2 and interleukin-4 receptors studied by molecular modelling.

BACKGROUND: Interleukin-2 (IL2) and interleukin-4 (IL4) are members of the four-helix bundle family of cytokines, whose receptors show similarity to each other and to the growth hormone receptor fold. These proteins help to control, among other things, the rate of clonal expansion of lymphocytes, and thus play an important role in the regulation of the immune system. They are therefore of interest as transmembrane signalling proteins, as well as potential pharmaceutical targets. RESULTS: We have modelled structures of the extracellular components of the IL2 and IL4 receptors based on the structure of the complex of human growth hormone with its receptor, and incorporating the recently discovered shared gamma c chain. The models provide possible explanations for several experimental observations, including those from site-directed mutagenesis around the binding sites. Receptor residues that may be close to important side chains on IL2 and IL4 are identified and possible effects of their mutation are discussed. A comparison is made between the models and the growth hormone complex, and between the gamma c chain bound to IL2 and to IL4. CONCLUSIONS: The models offer structural explanations for observed behaviour such as the effects of mutation of the A- and D-helices of the cytokines. In addition, they may be of use in the identification of residues which may interact in the ligand-receptor interfaces, and which would therefore be worthy of further investigation.

A computer model of the interleukin-4/receptor complex.

Interleukin-4 is a member of the cytokine family, a group of related messenger proteins which collectively help to moderate and control the immune response. It is believed that the folding topology of the beta-sheets of the interleukin-4 receptor (IL4R) is the same as that seen in the crystal structure of CD4. Although the sequence identity is low, homology modeling techniques have been used to model the IL4R structure from CD4. Refinement by molecular dynamics leads to a suggested structure which has been docked to interleukin-4 (IL4). Several residues of apparent importance for binding are identified.

Epithelial cell-fibroblast interactions in lung injury and repair.

Although direct intercellular contacts between alveolar epithelial cells and fibroblasts have been described in developing and adult lung, the frequency of such contacts and their relationship to type 2 cell division and differentiation in normal and abnormal repair is not known. The authors now correlate measurements of type 2 cell basal surface, basement membrane continuity, and the incidence of epithelial-interstitial cell contacts with the proliferative index of type 2 cells and fibroblasts in normal repair (after hyperoxia) and in abnormal repair with fibrosis (after bleomycin or butylated hydroxytoluene). In each case, type 1 cell necrosis was followed by an increase in type 2 cell basal surface as the cells spread over the denuded capillary wall before dividing. After hyperoxia, a high but short-lived peak in type 2 cell division was not accompanied by fibrosis. After more severe drug-induced injury, the type 2 proliferative phase was extended and was accompanied by prolonged fibroblast growth. Type 2 cells persisted where they covered a thick interstitium of fibroblasts and fibrillar collagen. The incidence of epithelial-interstitial cell contacts decreased at the time of maximal type 2 cell division, then increased immediately after the peak. The results suggest a reciprocal epithelial-fibroblast control system whereby 1) epithelial necrosis and delayed repair promotes fibroblast growth, and 2) direct contact of epithelial cells with fibroblasts or fibrillar collagen may provide a factor important for the regulation of type 2 cell growth and differentiation.

Silica-induced pulmonary fibrosis involves the reaction of particles with interstitial rather than alveolar macrophages.

Macrophage-derived products have been implicated in fibroblast stimulation following particle deposition in the lung. To assess the role of macrophages in the alveolus versus those in the interstitium in the induction of pulmonary fibrosis, we compared the pulmonary response to silica when phagocytosis occurred predominantly in each of these compartments. One group of mice received intratracheal silica which was phagocytosed largely by alveolar macrophages (AM). A second group was exposed to whole body irradiation prior to receiving the same dose of silica. This prevented the usual efflux of PMN and monocytes into the air sacs, allowing passage of silica particles across the alveolar epithelium to reach the interstitial macrophages (IM). In the irradiation plus silica group, many large interstitial granulomas were formed at 2-4 weeks, and collagen levels were significantly greater than in all other groups at 16 weeks. More silica was found in a lung tissue residue and in lymph nodes of these animals. Pulmonary fibrosis was limited to interstitial areas where there was a high level of retained silica, whereas peripheral regions of the lung, where free AM containing silica were found, did not show fibrosis of the alveolar walls. The results suggest that factors secreted by IM in response to silica are more effective in stimulating fibrogenesis than secretions made by the AM into the alveolar space.

Stereochemistry of valine biosynthesis. Configuration of the product of rearrangement of alpha-acetolactate.

When alpha-aceto[1,3,5-13C3]lactate (2-hydroxy-2-methyl-3-oxo[1,3,5-13C3]butanoate) was incubated with a cell-free system prepared from Salmonella typhimurium, the valine produced was labelled in the C-4 pro-S position. This result proves that during the tertiary ketol rearrangement catalysed by the reductoisomerase of the isoleucine-valine pathway, the methyl group transfer is to the re face of the trigonal centre at C-3 of alpha-acetolactate.