Exogenous bFGF promotes articular cartilage repair via up-regulation of multiple growth factors.

OBJECTIVE: To investigate the roles of exogenous basic fibroblast growth factor (bFGF) on the repair of full-thickness articular cartilage defects in rabbits. DESIGN: In the present study, a double-layered collagen membrane sandwiched with bFGF-loaded-nanoparticles between a dense layer and a loose layer was implanted into full-thickness articular cartilage defects in rabbits. By grafting the membrane in a different direction, the dense layer or the loose layer facing the surface of the subchondral bone, the effects of the released bFGF on the defects and the profiles of nine growth factors (GFs) in synovial fluid (SF) were investigated using histological methods and antibody arrays, respectively. RESULTS: In the group with the loose layer facing the surface of the subchondral bone, fast release of bFGF was observed, and early high levels of endogenous transforming growth factor-ß2 (TGF-ß2), vascular endothelial growth factor (VEGF), bFGF, bone morphogenetic protein 2 (BMP-2), BMP-3, and BMP-4 in SF were detected by antibody arrays, especially on day 3. Chondrocyte-like cells were also observed in this group at an early stage. As a result, this group showed better levels of repair, as compared to the other groups in which low GF levels were detected at an early stage, and chondrocyte-like cells appeared much later. CONCLUSIONS: Our study suggests that exogenous bFGF promotes articular cartilage repair by up-regulating the levels of multiple GFs, but administration at an early stage is required.

Interferon-beta reduces the mouse liver fibrosis induced by repeated administration of concanavalin A via the direct and indirect effects.

Type I interferons (IFNs), IFN-alpha and IFN-beta, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0.3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN-beta pretreatment. IFN-beta suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN-beta reduced the expressions of transforming growth factor-beta, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN-beta reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

Comparative effect of seeds of Rhynchosia volubilis and soybean on MG-63 human osteoblastic cell proliferation and estrogenicity.

The seeds of Rhynchosia volubilis (SRV) (Leguminosae) and soybean have been used in oriental folk medicine to prevent postmenopausal osteoporosis. Their beneficial effects are caused by a high content of isoflavone, which function as partial agonists or antagonists of estrogen. To compare the estrogenic effects of SRV and soybean on the MG-63 osteoblastic cell proliferation, 70% methanol extracts of SRV or soybean were treated on MG-63 cells. Although biphasic over a concentration range of 0.001 mg/ml-0.1 mg/ml, both SRV and soybean extracts increased MG-63 cell proliferation. However SRV was more effective at increasing the cell proliferation that paralleled with the greater estrogenic effects as determined by estrogen receptor alpha (ERalpha) expression, an estrogenic response element (ERE)-luciferase activity and the selective expression of insulin-like growth factor-I (IGF-I). SRV-induced IGF-I expression resulted from increases in the mRNA levels. Despite the increased expression of ERbeta, ERE activity and IGF-I expression by soybean were lower than those by SRV. Furthermore, the comparable estrogenic effects between SRV and the combined treatment of genistein and daidzein standards at 0.5 x 10(-8) M, which is a concentration of these two isoflavones similar to that of SRV at 0.001 mg/ml, demonstrate that the greater estrogenicity of SRV for MG-63 cell proliferation is mediated by the synergism of low levels of isoflavones for the selective expression of IGF-I.

A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke.

Oxidative stress is a major source of injury from cerebral ischemia and reperfusion. We hypothesized that a catalytic antioxidant AEOL 10150 [manganese (III) meso-tetrakis (di-N-ethylimidazole) porphyrin] would attenuate changes in brain gene expression in a mouse model of transient middle cerebral artery occlusion (MCAO). C57BL/6J mice were subjected to either sham surgery or 60 min of right MCAO. AEOL 10150 or phosphate-buffered saline was given intravenously 5 min after onset of reperfusion (n = 6 per group). Six hours later, parenchyma within the MCA distribution was harvested. RNA from the six brains in each group was pooled and mRNA expression determined using an Affymetrix murine MG_U74A v. 2.0 expression microarray. Each experiment was performed three times. The largest changes in expression occurred in stress response and inflammatory genes such as heat shock protein, interleukin-6, and macrophage inflammatory protein-2. Treatment with AEOL 10150 attenuated only the increase in expression of inflammatory genes. This suggests that AEOL 10150 protects brain by attenuating the immune response to ischemia and reperfusion.

Induction of granulin precursor gene expression by estrogen treatment in neonatal rat hypothalamus.

Our previous research has demonstrated that androgen treatment during the perinatal period increases granulin (grn) precursor mRNA levels in the neonatal rat hypothalamus. To elucidate whether exogenous estrogen increases grn mRNA in the neonatal hypothalami, expression of grn gene in the neonatal hypothalamus was studied by the competitive reverse transcription-polymerase chain reaction method. At 6 and 10 days of age, grn gene expression was significantly increased in the hypothalamus of pups whose dam has been dietarily administrated ethinyl estradiol from day 15 of gestation to the day of sampling. The subcutaneous injection of estradiol benzoate to neonatal rats at 2 days of age significantly increased grn gene expression on day 10. It was shown that estrogen, as well as androgen, was able to induce grn gene expression in the neonatal hypothalamus.

Oxidative stress, growth factor production and budding in potato tubers during cold storage.

In order to verify the role played by oxidation in the budding of potato tubers (Solanum tuberosum L. cv. Kennebec), the physiological events occurring below bud at 4 degrees C have been studied for a period of 6 months. The low temperature storage induced an increase in the degree of unsaturation and a decrease in the ratio of saturated/unsaturated fatty acids of membrane polar lipids with a subsequent increase of lipid hydroperoxides (LOOH). Cold stress increased both enzymatic antioxidative activities (superoxide dismutase, SOD, E.C.1.15.1.1; catalase, CAT, E.C.1.11.1.6), and alpha-tocopherol levels thus protecting membrane's polyunsaturated lipids. Between 0 and 15 days of storage SOD/CAT ratio, alpha-tocopherol, LOOH levels and the degree of lipid unsaturation showed strong variations. After 30 to 120/150 days the antioxidative system seemed to reach a homeostasis different from that of time 0, accompanied by a constant increase of indole-3-acetic acid (IAA) after 60 days. The antioxidative system, after 150 days, lost its efficiency while LOOH levels were maintained higher than time 0 and IAA concentration was sufficient to allow sprouting.

cDNA cloning of bovine midkine and production of the recombinant protein, which affects in vitro maturation of bovine oocytes.

In the present study, we cloned bovine midkine (bMK) cDNA by RT- and RACE-PCR, and determined its nucleotide sequence. The nucleotide and deduced amino acid sequences of bMK showed a high degree of homology to those of mouse and human MK. Moreover, a large amount of recombinant bMK (rbMK) was produced using a baculovirus expression system and the protein was purified to homogeneity by heparin affinity chromatography. To examine whether treatment with rbMK during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine cumulus-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 hr in IVM medium without (control) or with various concentrations (50-400 ng/ml) of rbMK, followed by in vitro fertilization (IVF) and culture. Although rbMK treatment during IVM did not affect the rates of nuclear maturation and postfertilization cleavage of oocytes, rbMK at all experimental concentrations significantly (P < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared to the control. Next, it was examined whether heparitinase (HTN) treatment of cumulus-enclosed oocytes would affect the enhancing activity of rbMK during IVM on the developmental competence of oocyte after IVF. The enhancing activity of rbMK was completely suppressed by HTN (1.0 mU/ml) treatment. These results indicate that the presence of rbMK during IVM of bovine cumulus-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that heparan sulfate chains on the cell surface of cumulus cells and/or oocytes are required for bMK to exert its effect.

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells.

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.

Possible molecular mechanisms of cryoablation-induced impotence in a rat model.

OBJECTIVES: Cryoablation of the prostate has been reported to induce impotence as a consequence of cavernosal nerve injury. This study is designed to investigate the early and late effects of cavernosal nerve cryoablation on growth factor expression and erectile function in a rat model. METHODS: Forty male rats were divided into two groups (n=20 each). The first group underwent unilateral cavernosal nerve freezing (experimental group). Before their euthanization at 1 and 3 months (10 rats each), erectile function was assessed by electrostimulation of the cavernous nerves. The second group served as the control and was killed at the same time points. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to identify protein and gene expression of nerve growth factor (NGF), transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) in the rat penis and pelvic ganglia. RESULTS: Electrostimulation of the frozen nerve after 3 months revealed a significantly higher maximal intracavernosal pressure and a shorter latency period than in the 1-month group. At 3 months, immunoblot showed upregulation of NGF, TGF-alpha, and the precursor form of IGF-1 protein expression in the penile tissue; RT-PCR showed downregulation of NGF gene expression in the pelvic ganglia of the frozen side. CONCLUSIONS: The results show that erectile function decreased at 1 month and then partially recovered 3 months after cavernosal nerve freezing. This alteration in erectile function was associated with differential gene and protein expression of the growth factors (NGF, TGF-alpha, EGF, and IGF-1). Further studies are required to elucidate the potential role of these growth factors in the prevention and treatment of cryoablation-induced impotence.

Reynosin from Sassurea lappa as inhibitor on CINC-1 induction in LPS-stimulated NRK-52E cells.

An inhibitor on CINC-1 (cytokine-induced neutrophil chemoattractant-1) induction in LPS-stimulated rat kidney epithelioid NRK-52E cells was purified from the roots of Sassurea lappa Clarke, a herbal medicine used in Korean traditional prescriptions for gastric intestinal diseases by a variety of column chromatographic procedures. The inhibitor was identified as reynosin, a sesquiterpene lactone isolated and characterized previously from Ambrosia confertiflora DC., and Magnolia grandiflora L. Reynosin exhibited a dose-dependent inhibition on CINC-1 induction in LPS-stimulated NRK-52E cells, where 50% of inhibitory effect was shown at the concentration of about 1 microM.

Hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary inflammation following lipopolysaccharide.

Recent studies have suggested that hemorrhagic shock followed by resuscitation renders patients more susceptible to lung injury by priming for an exaggerated response to a second stimulus, the so-called "two-hit" hypothesis. We investigated the role of C-X-C chemokines in mediating the augmented lung inflammation in response to LPS following resuscitated shock. In a rodent model, animals exposed to antecedent shock exhibited enhanced lung neutrophil sequestration and transpulmonary albumin flux in response to intratracheal LPS. This effect correlated with an exaggerated expression of cytokine-induced neutrophil chemoattractant (CINC) protein and mRNA, but not macrophage-inflammatory protein 2. Strategies designed to inhibit CINC, both anti-CINC Ab and supplementation with the antioxidant N-acetyl-cysteine, prevented the enhanced neutrophil sequestration, suggesting that CINC played a central role in the enhanced leukocyte accumulation following shock plus LPS treatment. Shock alone increased lung nuclear factor-kappaB expression and augmented the response to LPS. Prevention of this effect by N-acetylcysteine supplementation of the resuscitation fluid implicates a role for oxidant stress in the priming for lung inflammation following shock. Finally, alveolar macrophages recovered from shock-resuscitated animals released more CINC protein in vitro in response to LPS than macrophages from sham animals. Considered together, these findings show that augmented release of CINC, in part from primed alveolar macrophages, contributes significantly to the enhanced lung leukosequestration and transpulmonary albumin flux in response to LPS following resuscitated shock.

Expression of activin and follistatin in the rat hypothalamus: anatomical association with gonadotropin-releasing hormone neurons and possible role of central activin in the regulation of luteinizing hormone release.

The central role of activin in the regulation of the reproductive axis remains largely unexplored. Evidence suggests that activin may play a role in control-linggonadotropin-releasing hormone (GnRH) release. We assessed potential neuroanatomical associations between activin- and GnRH-neuronal systems via examination of the distribution of activin betaA-subunit and activin binding protein (follistatin) protein and mRNA signals relative to GnRH neurons in the adult rat brain. Activin betaA-subunit-immunostained fibers were distributed throughout the hypothalamus and GnRH-positive perikarya, and fibers were in close association with betaA-subunit-immunoreactive fibers. Follistatin mRNA-expressing cells were also identified throughout the hypothalamus with GnRH fibers often observed juxtaposed to follistatin cell bodies. Colocalization of either the betaA-subunit or follistatin within GnRH neurons was not detected. The functional significance of central activin in the regulation of the reproductive axis was also demonstrated. The intracerebroventricular infusion of rh-activin A significantly increased luteinizing hormone, but not follicule-stimulating hormone, serum levels in adult male rats. Taken together, the present results support an interaction between activin and GnRH neuronal systems in the rat hypothalamus, and suggest activin may act within the brain to regulate the reproductive axis.

Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus.

Hepatoma-derived growth factor (HDGF) is an acidic polypeptide with mitogenic activity for fibroblasts performed outside the cells despite the presence of a putative nuclear localization signal (NLS). We have now cloned three related mouse cDNAs: one for a mouse homologue of human HDGF and two for additional HDGF-related proteins provisionally designated HDGF-related proteins 1 and 2 (HRP-1 and -2). Their deduced sequences have revealed that HDGF belongs to a new gene family with a highly conserved 98-amino-acid sequence at the amino terminus (hath region, for homologous to the amino terminus of HDGF). HRP-1 and HRP-2 proteins are 46 and 432 amino acids longer than mouse HDGF, respectively, and have no conserved amino acid sequence other than the hath region. HRP-1 is a highly acidic protein (26% acidic) and also has a putative NLS. HRP-2 protein carries a mixed charge cluster, a sharp switch of positive-to negative-charge residues, which is often found in some nuclear proteins. Northern blotting shows that mouse HDGF and HRP-2 are expressed predominantly in testis and skeletal muscle, to intermediate extents in heart, brain, lung, liver, and kidney, and to a minimal extent in spleen. HRP-1 is expressed specifically in testis. These findings suggest that the HDGF gene family might play a new role in the nucleus especially in testis.

Enhanced expression of nucleobindin in lymphatic organs of lupus-prone mice.

Nucleobindin (Nuc) was originally identified as a 55 kDa protein that enhanced anti-DNA antibody production in cultures of autoimmune MRL/lpr mouse spleen cells. cDNA cloning and the production of recombinant protein (rNuc) have revealed that rNuc binds to DNA and Ca2+ by means of leucine zipper and EF-hand motif structures. In vitro and in vivo effects of rNuc to induce autoantibodies including anti-dsDNA antibody in normal mice have been demonstrated; however, whether Nuc is involved in the state of autoimmunity occurred in MRL/lpr, C3H/gld and male BXSB mice has not been elucidated. Here we report that the expression of Nuc mRNA is upregulated with age in the lymphatic organs and is positively correlated to the serum levels of Nuc in these lupus-prone strains of mice. Upon immunization with KLH in Freund's complete adjuvant, normal BALB/c mice also showed an elevated level of Nuc in their serum and elevated Nuc mRNA in their lymphatic organs. In addition, in vitro stimulation of BALB/c splenocytes with Con A or LPS remarkably enhanced Nuc mRNA expression. These results suggest that upregulation of Nuc mRNA in lymphatic organs and serum Nuc of lupus-prone mice is related to spontaneous activation of immunocompetent cells; the present data are also consistent with our previous hypothesis on the role of Nuc in the induction or enhancement of autoimmunity in lupus models of mice.

A possible growth factor role of IL-6 in neuroectodermal tumours.

Preliminary data have shown that IL-6 may act as an autocrine growth factor to control proliferation. We further characterised the role of IL-6 in tumour growth as an autocrine/paracrine growth factor in neuroectodermal tumours. We evaluated the production and secretion of IL-6 by seven human melanoma, five neuroblastoma and one glioblastoma cell lines. Moreover, we determined their IL-6-dependent growth in serum free-medium or under minimal growth-supplement conditions: IL-6 dependent growth was observed in two non-IL-6 producing melanoma and in one neuroblastoma cell lines. In addition, expression of IL-6 mRNA and peptide was increased by retinoic acid. The data support the hypothesis that IL-6 contributes to neuroectodermal tumour growth, even though it shows a less potent effect than other reported growth factor such as IGF-II.

Cytokine-induced neutrophil chemoattractant gene expression in the rat hypothalamus by osmotic stimulation.

Cytokine-induced neutrophil chemoattractant (CINC) is one of the chemokines and has chemotaxity for neutrophils. Recently, we found the presence of stress-sensitive CINC expression in the hypothalamic nuclei such as the paraventricular nucleus. Since CINC was predominantly co-localized with vasopressin in the supraoptic nucleus (SON), we investigated the effect of hyperosmotic challenge on CINC mRNA in the hypothalamus. We found that CINC mRNA expression in the hypothalamus was augmented within 30 min following osmotic stimulation and immediately returned to the basal level. The suckling, which is a stimulation to oxytocin neurons in the SON, has no effect on CINC mRNA expression in the hypothalamus. This is the first evidence that the chemokine in the brain is activated by osmotic stimulation.

Human intestinal trefoil factor is expressed in human hypothalamus and pituitary: evidence for a novel neuropeptide.

Human intestinal trefoil factor, hITF, a secretory polypeptide found mainly in the human gastrointestinal tract, is a member of the newly characterized trefoil factor or P-domain peptide family representing putative growth factors. Here we describe the identification of this gut peptide in the human brain and pituitary. With reverse transcriptase polymerase chain reaction, we were able to isolate and clone the transcript from human hypothalamus. An antibody generated against a synthetic peptide derived from the carboxyl terminus of hITF was used for immunohistochemical studies of appropriate tissue sections. Neurons expressing hITF were identified in two magnocellular hypothalamic nuclei, the paraventricular and periventricular nuclei. hITF polypeptide was also observed in Herring bodies of the neurohypophysis and in secretory cells of the adenohypophysis. Double immunostaining with antigrowth hormone antibody showed partial coexistence in a selected subpopulation of adenohypophysial cells. Localization of hITF in the hypothalamo-neurohypophysial system may suggest a modulatory action on the classical magnocellular nonapeptides vasopressin and oxytocin, and further indicates an adenohypophysial importance of this peptide. It is likely that hITF represents a novel neuropeptide of yet unknown function.

In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation.

We hypothesized that endotoxin injection in rats would stimulate in vivo nuclear factor-kappa B (NF-kappa B) activation in lung tissue and that antioxidant treatment before endotoxin injection would attenuate endotoxin-induced NF-kappa B activation, chemokine gene expression, and neutrophilic lung inflammation. We studied NF-kappa B activation in rat lung tissue following a single i.p. injection of endotoxin (6 mg/kg). After in vivo endotoxin treatment, lung NF-kappa B activation peaked at 2 h and temporally correlated with the expression of cytokine-induced neutrophil chemoattractant mRNA in lung tissue. Treatment with the antioxidant N-acetylcysteine (NAC) 1 h before endotoxin resulted in decreased lung NF-kappa B activation in a dose-dependent manner (from 200-1000 mg/kg) and diminished cytokine-induced neutrophil chemoattractant mRNA expression in lung tissue. Treatment with NAC significantly suppressed endotoxin-induced neutrophilic alveolitis. The average total lung lavage neutrophil count was 5.5 x 10(6) with endotoxin treatment vs 0.9 x 10(6) with NAC treatment before endotoxin. The NF-kappa B pathway represents an attractive therapeutic target for strategies to control neutrophilic inflammation and lung injury.

Interaction of the protein nucleobindin with G alpha i2, as revealed by the yeast two-hybrid system.

The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.

Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis.

The present study was undertaken to further define the role of alveolar macrophages (AM) in the pulmonary response to crocidolite fibers. Briefly, groups of 4 male F344 rats were intratracheally instilled with saline or saline suspensions of crocidolite at 2 or 20 mg/kg body weight. Animals were sacrificed 3, 7, 14, and 28 d after exposure and the lung response was characterized by analysis of bronchoalveolar lavage fluid (BALF) for markers of lung injury and inflammation. AM obtained in BALF were cultured and their production of the pro-inflammatory cytokines, tumor necrosis factor alpha (TNF alpha), and interleukin-1 (IL-1) were characterized along with fibronectin, a protein known to stimulate fibroblast migration and proliferation. Lung hydroxyproline content was determined 28 d after exposure and lung histopathology was characterized on d 28 and 90 after exposure. Crocidolite instillation resulted in transient dose-related pulmonary inflammation as evidenced by increased numbers of BALF neutrophils at the low dose and neutrophils, macrophages, and lymphocytes at the high dose. Cytotoxicity and increased permeability were demonstrated by increased levels of BALF lactate dehydrogenase (LDH) and total protein, respectively. AM TNF alpha and IL-1 production were increased only at the high crocidolite dose. This cytokine response was greatest at d 3 and decreased thereafter. AM TNF alpha and IL-1 release were positively correlated with the increased BALF neutrophils. In contrast to TNF alpha and IL-1, AM fibronectin release was increased at both the low and high doses, with the magnitude of response increasing over time. Consistent with previous acute asbestos inhalation studies, histopathology revealed inflammation localized at the level of the terminal bronchioles and alveolar ducts. Fibrosis was demonstrated at both doses by increased trichrome staining of lung tissue sections. Only the high dose resulted in a detectable increase in lung hydroxyproline. Given the bioactivities of TNF alpha, IL-1, and fibronectin, their increased production after crocidolite exposure indicates they contribute to the pulmonary inflammation and fibrosis occurring with this mineral fiber. In addition, the correlation of increased AM TNF alpha and IL-1 production with increased BALF neutrophils supports a role for these cytokines in crocidolite-induced inflammatory cell recruitment. Lastly, association of a persistent increase in AM fibronectin production with an eventual increase in lung collagen deposition extends the growing database indicating this response is a predictive marker of pulmonary fibrosis.