Prostaglandin protection of human isolated gastric glands against indomethacin and ethanol injury. Evidence for direct cellular action of prostaglandin.

Isolated human gastric glands from surgical specimens were preincubated in an oxygenated medium with placebo or 16,16 dimethyl prostaglandin E2 (dmPGE2) and incubated at 37 degrees C in either medium alone, medium containing 4.43 mM indomethacin or medium containing 8% ethanol. We assessed the viability of gland cells with fast green exclusion, release of lactate dehydrogenase (LDH) into the medium, and ultrastructural damage by scanning and transmission electron microscopy. Both indomethacin and ethanol significantly reduced the viability of placebo-pretreated glands, increased LDH release into the medium, and produced prominent ultrastructural damage. DmPGE2 significantly reduced both indomethacin and ethanol-induced injury, increased the number of viable cells, reduced LDH release, and diminished the extent of ultrastructural damage. These studies indicate that PG protection of gastric mucosal cells has a direct cellular action that is not limited to replacement of depleted endogenous PGs. PG protection in our experiments did not depend on PG's previously described systemic actions, such as protection of the microvessels, preservation of the mucosal blood flow, or stimulation of bicarbonate and mucus secretion.

Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes.

Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.

Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo.

Keratinocyte growth factor (KGF) administered as a single intratracheal injection causes a prominent dose-dependent proliferation of type II alveolar epithelial cells in the lungs of adult rats. The increase in mitotically active alveolar cells histologically appears as a micropapillary epithelial cell hyperplasia after 2 d and peaks after 3 d in the form of monolayers of cuboidal epithelial cells lining alveolar septae. Proliferating cell nuclear antigen immunohistochemistry confirmed the profound proliferative response induced by KGF. The hyperplastic alveolar lining cells contain immunoreactive surfactant protein B and are ultrastructurally noted to contain lamellar inclusions characteristic of surfactant-producing type II pneumocytes. Mild focal bronchiolar epithelial hyperplasia is noted but is much less striking than the proliferation of type II pneumocytes. Large airways are unaffected by KGF. Daily intravenous injection of KGF is also able to cause pneumocyte proliferation. The normal adult rat lung constitutively expresses both KGF and KGF receptor mRNA, suggesting that endogenous KGF may be implicated in the paracrine regulation of the growth of pneumocytes. In conclusion, KGF rapidly and specifically induces proliferation and differentiation of type II pneumocytes in the normal adult lung.

Keratinocyte growth factor ameliorates cyclophosphamide-induced ulcerative hemorrhagic cystitis.

To determine whether keratinocyte growth factor (KGF), an epithelial and urothelial growth factor, ameliorates cyclophosphamide (CP)-induced cystitis in rats, KGF (5 mg/kg) was injected in rats as a single i.v. injection 24 h prior to i.p. injection of CP (200 mg/kg). Bladders were evaluated histologically 48 h after CP injection, and KGF pretreatment was found to almost completely prevent CP-induced ulcerative hemorrhagic cystitis. Urinary KGF levels were measured by ELISA, and KGF was found to be undetectable in control urine, but it was found to appear in the urine of KGF-treated rats at 8 h, with a peak concentration of approximately 10 ng/ml. Bilateral nephrectomy did not diminish the proliferative effect of KGF on urothelium, suggesting that the contribution of urinary KGF to urothelial proliferation is insignificant. In conclusion, systemic administration of KGF is protective against CP-induced cystitis. Although KGF appears in the urine, urinary KGF is not necessary for the proliferative action of KGF on urothelium.

Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Effect of IgG subclasses on in vivo bioavailability and metabolic fate of immune-complexed insulin in Lewis rats.

The bioavailability, distribution, and metabolic fate of 125I-labeled insulin complexed to antibodies in guinea pig antiserum, purified guinea pig IgG1, IgG2, a mixture of IgG1 and IgG2, and homologous Lou/m rat antiserum were studied in inbred Lewis rats. 125I-insulin complexed to purified guinea pig IgG2 antibodies was rapidly cleared from the blood and sequestered in increasing amounts with time in the liver. Large amounts of the 125I-insulin complexed to guinea pig IgG1 antibodies remained in the blood for at least 30 min. The bioavailability of 125I-insulin bound to IgG1 and IgG2 antibodies was inhibited for at least 30 min because significantly less was available for rapid binding to insulin receptors on hepatocytes and renal tubular cells and its subsequent rapid degradation. The bioavailability of 125I-insulin was further decreased when bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies compared with the 125I-insulin complexed to either purified IgG1 or IgG2 antibodies alone. The 125I-insulin bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies was distributed in vivo in a manner reflecting the relative concentrations of the IgG1 and IgG2 antibodies present. The bioavailability, distribution, and metabolic fate of 125I-insulin in immune complexes prepared with homologous Lou/m rat insulin antiserum was qualitatively similar to that observed with immune complexes prepared with guinea pig insulin antiserum. It appears that the Lewis rat can be used as an in vivo model to study the bioavailability,distribution,and metabolic fate of insulin bound to xenogenic or homologous insulin antibodies.

Hematologic interactions of endotoxin, tumor necrosis factor alpha (TNF alpha), interleukin 1, and adrenal hormones and the hematologic effects of TNF alpha in Corynebacterium parvum-primed rats.

Endotoxin reduces the release among other cytokines of tumor necrosis factor (TNF) and interleukin 1 (IL-1) and causes peripheral lymphopenia and a dose-response-dependent initial neutropenia followed by a monophasic neutrophilia. TNF alone induces lymphopenia and an initial neutropenia followed by a biphasic neutrophilia. IL-1 alone induces lymphopenia and a monophasic neutrophilia. TNF-plus-IL-1 caused a greater lymphopenia than either monokine alone, suggesting that both monokines contribute to LPS-induced lymphopenia. TNF-plus-IL-1 induced neutropenia similar in magnitude to that induced by TNF alone and induced a neutrophilia significantly greater than that induced by either monokine alone, suggesting that LPS-induced neutropenia is caused by TNF, while LPS-induced neutrophilia is due to the combined effects of TNF and II-1. TNF and IL-1 were administered together with LPS to simulate the in vivo condition of endogenous monokine release during gram-negative bacteremia. TNF combined with LPS increased both the duration and magnitude of LPS-induced lymphopenia, LPS-induced neutropenia, and LPS-induced neutrophilia. TNF-plus-LPS treated rats at 2 hours after injection exhibited a striking 93% decrease in bone marrow neutrophils even though no peripheral neutrophilia was yet apparent, suggesting that the subsequent neutrophilia was due to demargination and recirculation of neutrophils sequestered in the peripheral vasculature immediately after their release from the bone marrow. Epinephrine, which causes neutrophilia by demargination but not by release of marrow neutrophils, reversed the initial neutropenia in TNF-plus-LPS-treated rats and increased the neutrophilia. IL-1 combined with LPS increased LPS-induced neutrophilia, suggesting that endogenous IL-1 also contributed to LPS-induced neutrophilia. Corynebacterium parvum-primed rats with hyperplasia of the monocyte-macrophage system and treated with TNF differed from naive rats treated with TNF in that the second peak was as great as the initial peak of neutrophilia, supporting the hypothesis that the second peak of TNF-induced neutrophilia is due to the release of endogenous monokines. In conclusion, exogenous TNF, IL-1, and adrenal hormones affect circulating numbers of lymphocytes and neutrophils in a fashion consistent with their postulated endogenous role in the regulation of leukocyte trafficking during bacterial infection.

The contributions of adrenal hormones, hemodynamic factors, and the endotoxin-related stress reaction to stable prostaglandin analog-induced peripheral lymphopenia and neutrophilia.

Stable prostaglandin analogs are known to induce lymphopenia and neutrophilia in a dose-dependent fashion after subcutaneous injection in rats. The purpose of the present investigation is to determine whether the prostaglandin-induced changes in circulating leukocytes might be secondary to hypotension with the ensuing release of adrenal hormones. The adrenal medullary catecholamine epinephrine was found to induce neutrophilia in both intact and adrenalectomized rats, and the glucocorticosteroid analog dexamethasone induced a profound lymphopenia in rats as reported by previous investigators. A stable analog of PGF2 alpha (15-S-15-methyl PGF2 alpha; M-PGF2 alpha) at the dose of 1 mg/kg induced marked systemic hypotension 1 h after injection, with lymphopenia and neutrophilia 6 h after injection. The non-prostanoid hypotensive agent captopril, at a dose of 63 mg/kg, induced a hypotension of similar magnitude and kinetics to that induced by prostaglandin. Captopril also induced lymphopenia and neutrophilia at 6 h, although the neutrophilia was of lesser magnitude than that induced by prostaglandins. The prostaglandin-induced lymphopenia was found to be mediated, at least in part, by the hypotension-induced release of adrenal hormones, as evidenced by the abrogation of lymphopenia in prostaglandin-treated adrenalectomized rats. Captopril-treated adrenalectomized rats, however, did develop a significant lymphopenia, suggesting that hypotension can result in lymphopenia even in adrenalectomized rats. The M-PGF2 alpha-induced neutrophilia in adrenalectomized rats, by comparison to captopril-induced neutrophilia in adrenalectomized rats, was greater than the neutrophilia expected as the result of hypotension alone. Indeed, the M-PGF2 alpha-induced neutrophilia in adrenalectomized rats was greater than the captopril-induced neutrophilia in sham-adrenalectomized rats. Thus, a portion of the neutrophilia induced by M-PGF2 alpha in intact rats may be mediated through adrenal-independent, hemodynamic-independent mechanisms. The possibility that M-PGF2 alpha might be inducing neutrophilia via an endotoxin-like stress reaction was investigated by examining changes in circulating white blood cells in intact and adrenalectomized C3H/HeN (endotoxin-sensitive) and C3H/HeJ (endotoxin-resistant) mice after prostaglandin administration. No quantitative differences in the prostaglandin-induced neutrophilia were noted in C3H/HeJ mice as compared to the C3H/HeN mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of tumor necrosis factor alpha-induced lymphopenia, neutropenia, and biphasic neutrophilia: a study of lymphocyte recirculation and hematologic interactions of TNF alpha with endogenous mediators of leukocyte trafficking.

Tumor necrosis factor alpha (TNF) induces lymphopenia, neutropenia, and biphasic neutrophilia after intravenous injection of 3,000 U TNF in Lewis rats. The mechanism of TNF-induced lymphopenia was investigated by means of thoracic duct cannulation. Hourly measurements of lymphocyte recirculation via the thoracic duct failed to reveal any significant decrease in lymphocyte recirculation in TNF-treated vs. control rats, suggesting that a decrease in lymphocyte recirculation through the thoracic duct is not the mechanism for TNF-induced lymphopenia. The mechanism of TNF-induced neutropenia was investigated by administering TNF to rats in whom a neutrophilia had been induced with interleukin-1 (IL-1). In rats with neutrophilia, TNF resulted in a sharp decrease in the circulating neutrophil pool, demonstrating that TNF induces neutropenia by causing neutrophils to leave the circulating pool rather than decreasing neutrophil release from the marrow. The mechanism of neutropenia was furthermore shown to be due to the transient intravascular margination of neutrophils by administering epinephrine concomitantly with TNF. Epinephrine, which causes neutrophilia solely by demargination, abrogated the TNF-induced neutropenia and actually resulted in a neutrophilia that was greater than the neutrophilia occurring in epinephrine alone-treated rats, demonstrating both that TNF had already caused release of marrow neutrophils at the time of peripheral neutropenia, and that the paradoxical neutropenia was due to the transient intravascular margination of neutrophils. The known property of epinephrine to cause neutrophilia exclusively by demargination was proved by examination of the bone marrow of epinephrine-treated rats in whom no decrease in marrow neutrophils was observed (in contrast to TNF- and IL-1-treated rats in whom neutrophilia is accompanied by a depletion of marrow neutrophils). The mechanism of TNF-induced neutrophilia was investigated by modulating the magnitude of both the first and second peaks of neutrophilia by priming of rats with daily injections of IFN gamma for 2 days prior to administration of TNF. The first peak of neutrophilia in IFN gamma-primed TNF-treated rats was decreased in comparison to TNF alone-treated rats because of the well-known neutropenic and myelosuppressive effect of IFN gamma, which resulted in a decrease in the number of neutrophils that could be recruited to cause neutrophilia.(ABSTRACT TRUNCATED AT 400 WORDS)

The hematopoietic and mature blood cells of the rat: their morphology and the kinetics of circulating leukocytes in control rats.

Although the morphology of the hematopoietic and circulating blood cells of the rat differs slightly from that of human blood cells, the basophil is the only human blood cell for which the rat does not have a readily recognizable counterpart. The morphological classification of the rat's marrow and peripheral blood cells as based on our experience is described with reference to the differing observations of previous investigators and with comparison to the human hematopoietic system. Examination of the bone marrow and peripheral blood demonstrated that control rats injected i.v. with 1% normal rat serum in sterile saline exhibit a moderate transient lymphopenia as the only significant hematologic fluctuation induced by the stresses of injection, general anesthesia, and tail bleeding. Morphologic quantitation of marrow and peripheral blood cells complements the insights obtained by the culture of marrow cells. An appreciation of the morphology of the rat's blood cells and of the reproducible and quantitative nature of marrow and peripheral blood smear examinations may lead to more in vivo investigations of hematopoiesis and leukocyte trafficking.

Tumor necrosis factor exerts dose-dependent effects on erythropoiesis and myelopoiesis in vivo.

Recombinant human tumor necrosis factor alpha (TNF) administered i.v. to Lewis rats as a daily low dose for 1 week induces an erythroid hyperplasia of late normoblasts. Although the erythroid marrow compartment is hyperplastic, the morphology of the normoblasts is dysplastic and there is no accompanying increase in circulating red blood cells, suggesting a state of ineffective erythropoiesis. TNF administered on the same daily schedule as a high dose induces an erythroid hypoplasia of late normoblasts and a peripheral anemia with decreases in the hematocrit and hemoglobin. A tremendous myeloid hyperplasia is noted in rats treated with high-dose TNF, and the mechanism of the erythroid anemia may be in part due to the increase in neutrophils. In support of the hypothesis that the erythroid anemia may be partly myelophthisic in nature, a decrease in marrow lymphocytes was also noted. On the other hand, the dysplastic morphology of the late erythroid precursors in rats treated with low-dose TNF would also be consistent with a destructive effect of TNF on erythroid precursors as a mechanism of TNF-related anemia. In light of the in vitro inhibitory effects of TNF on erythropoiesis and myelopoiesis as reported by previous investigators, the erythroid and myeloid hyperplasia noted in vivo most likely represent indirect effects.

The erythropoietic effects of interleukin 6 and erythropoietin in vivo.

The purpose of this investigation was to compare the erythropoietic effects of recombinant interleukin 6 (IL-6) and recombinant erythropoietin (EPO) on the marrow and peripheral blood in vivo. IL-6 administered to rats as a single i.v. injection induces a selective erythroid hyperplasia of the marrow's late normoblasts at 12 and 24 h with a return to preinjection numbers of normoblasts at 48 and 72 h. The hyperplasia of late normoblasts in the marrow is accompanied by a left-shifted peripheral reticulocytosis. Daily injection of IL-6 does not induce any effects on the erythroid population of the marrow or circulation beyond those of a single injection. After daily administration of IL-6 for 4 or 7 days, the erythroid differential in the marrow and the peripheral reticulocyte count are equal to negative control values, indicating a rapid tachyphylaxis to the erythropoietic effect of IL-6. In contrast to IL-6, EPO administered as a single i.v. injection induces a panerythroid marrow hyperplasia with sequential peak increases in pronormoblasts and early normoblasts at 24 h, intermediate normoblasts at 24-48 h, and late normoblasts at 72 h. The peripheral reticulocyte count mirrors the development of erythroid precursors in the marrow by demonstrating an increasing left-shifted reticulocytosis between 24 and 72 h. Daily injection of EPO for 7 days induces a striking erythroid hyperplasia and a myeloid hypoplasia in the marrow. In summary, IL-6 in vivo is a differentiation factor that rapidly induces tolerance to its own effect, whereas EPO in vivo affects all stages of erythropoiesis and sustains erythropoiesis indefinitely. IL-6 may be one of the non-EPO factors in pokeweed mitogen spleen cell-conditioned medium that has been reported by previous investigators to enhance erythropoiesis, although many of those factors were thought to act upon an earlier stage of erythropoiesis. IL-6 is unlikely to exert an indirect erythropoietic effect in vivo via the induction of EPO because the sera of IL-6-treated rats did not contain elevated levels of EPO and because the effects of exogenously administered IL-6 and EPO are so different.

PEG-rHuMGDF promotes multilineage hematopoietic recovery in myelosuppressed mice.

PEG-rHuMGDF administered to normal mice is a lineage-specific growth factor for megakaryocytes and platelets as judged by morphologic examination of hematologic cells in marrow and peripheral blood smears. The purpose of this study was to document that PEG-rHuMGDF in myelosuppressed mice promotes multilineage hematopoietic recovery. High-dose 5-fluorouracil (5-FU) in mice results in profound myelosuppression and 0-30% survival. Mice receiving a single dose of PEG-rHuMGDF (1000 microg/kg) 1 day after 5-FU (225 mg/kg) demonstrate an increased survival (76% vs 27% in control mice at 14 days). Compared to surviving controls, PEG-rHuMGDF-treated mice not only show the expected higher platelet counts, but also increased marrow colony-forming unit granulocyte-macrophage, increased multilineage marrow cellularity, and increased neutrophil, monocyte, and lymphocyte counts in peripheral blood. PEG-rHuMGDF- and vehicle-treated mice both develop hepatic abscesses after 5-FU treatment, but the abscesses in the PEG-rHuMGDF-treated mice contain more neutrophils, suggesting that myeloid reconstitution contributes to their survival. Furthermore, survival in 5-FU-treated mice is significantly improved by granulocyte colony-stimulating factor and antibiotics, suggesting that infection rather than thrombocytopenia is the predominant cause of death. PEG-rHuMGDF after 5-FU promotes survival accompanied by accelerated lymphohematopoietic repopulation, suggesting that PEG-rHuMGDF, a lineage-specific thrombopoietic factor in normal mice, promotes multilineage hematopoietic recovery in myelosuppressed mice.

The prolonged hematologic effects of a single injection of PEG-rHuMGDF in normal and thrombocytopenic mice.

A single injection of > or =10 microg/kg PEG-rHuMGDF in mice causes a dose-dependent increase in circulating platelets beginning on day 3 and peaking on days 5-6. The mean platelet volume and platelet distribution width at doses > or =100 microg/kg initially increase in a dose-dependent fashion and later decrease. However, the mean platelet volume does not change when platelets are incubated with PEG-rHuMGDF in vitro. The number of marrow megakaryocytes increases in a dose-dependent fashion as early as day 1 and peaks on day 3. Marrow megakaryocyte colony-forming units (CFU-Meg) do not increase on days 1-3 at a dose of 100 microg/kg (a dose that increases platelet numbers two- to threefold and may be clinically relevant), but the relative frequency of high ploidy megakaryocytes and the proportion of large marrow megakaryocytes (29-50 microm in diameter) increases. After a dose of 1,000 microg/kg the percentage of megakaryocytes in mitosis peaks at 24-48 hours and the percentage of megakaryocytes incorporating BrdU is maximal at 48 hours, the relatively delayed peak of BrdU incorporation most likely representing endomitosis. The relative frequency of type II and III megakaryocytes peaks on days 3 and 4, respectively. Pharmacokinetic analysis of PEG-rHuMGDF shows peak serum concentrations at 2-4 hours and a terminal half-life of 11.4+/-2.5 hours. A single injection of PEG-rHuMGDF ameliorates carboplatin-induced megakaryocytopenia and thrombocytopenia in a dose-response dependent fashion. In conclusion, a single injection of PEG-rHuMGDF increases megakaryocyte and platelet production in normal and myelo-suppressed mice.

Chronic active hepatitis of hepatitis B and non-A, non-B etiology.

Sixty-six consecutive liver biopsies demonstrating chronic hepatitis (CAH) were stained for the presence of HBsAg using the three-step peroxidaseantiperoxidase technique. Only cases of CAH thought to be attributable to either hepatitis B or non-A, non-B hepatitis were included in this series. Twenty-three of 30 biopsies taken from 24 patients with HBsAg-positive serum stained positively. None of the HBsAg sero-negative cases stained postively. Hepatocytes staining positively for HBsAg were generally few in number and randomly distributed within the liver lobules. Three cases of membranous staining were noted. After grading both the degree of inflammatory activity and the amount of HBsAg staining, we found that a statistically significant inverse relationship exists. The biopsies of six of the GBsAg sero-positive patients who had received steroid therapy for their liver disease did not stain differently from the biopsies of the remaining 18 HGsAg sero-positive patients. Stains for HBsAg may help in distinguishing acute hepatitis B (HB) superimposed on preexisting liver disease from hepatitis B-CAH (CAHB). This distinction may be possible because tissue staining almost always is negative in acute HB, whereas it often is focally positive in CAHB. This application of immunoperoxidase may be especially useful in patients who are drug addicts.

Effect of dimethylthiourea on plasma paraquat concentration.

In a previous study, administration of the hydroxyl radical scavenger, dimethylthiourea (DMTU), and paraquat was associated with higher mortality in rats than was paraquat alone. In the present study, the possibility was evaluated that administration of DMTU increased plasma paraquat levels. Plasma paraquat concentrations were measured in Sprague-Dawley rats 1, 2, 4, 8 and 24 h after intraperitoneal (i.p.) injection of 29 mg paraquat cation/kg body wt. Another group of rats was treated identically except that they received i.p. injections of DMTU before injections of paraquat. Administration of DMTU was associated with increased plasma paraquat concentration (P less than 0.01). Pharmacokinetic analyses indicated that, compared to rats receiving paraquat alone, rats given paraquat and DMTU showed: (1) greater area under the paraquat concentration time-curve; (2) lower total body paraquat clearance; and (3) smaller apparent volume of distribution. Plasma biochemical studies indicated that paraquat caused hyperglycemia as well as an early reduction (compared to controls) in hepatic enzymes. We conclude that: (1) DMTU administration is associated with increased plasma paraquat concentrations; and (2) impaired synthesis or inhibition of release of hepatic proteins may be an early effect of paraquat.

Radiation-induced lung injury in vivo: expression of transforming growth factor-beta precedes fibrosis.

Cytokine release from irradiated cells has been postulated to start soon after irradiation preceding detectable clinical and pathological manifestation of lung injury. The expression of transforming growth factor beta (TGF beta), a fibrogenic and radiation-inducible cytokine, was studied from 1-16 weeks after the 15 and 30 Gray (Gy) of thoracic irradiation to rats. Thoracic irradiation caused an increase in TGF beta protein in bronchoalveolar lavage (BAL) fluid peaking at 3-6 weeks as compared to sham-irradiated control rats. Steady state TGF beta mRNA expression as shown by whole lung northern blot assay paralleled the TGF beta protein expression in BAL fluid. The peak of TGF beta protein increase in BAL fluid between 3 and 6 weeks coincided with the initial influx of inflammatory cells in BAL fluid, but preceded histologically discernable pulmonary fibrosis that was not apparent until 8-10 weeks after irradiation. In conclusion. TGF beta and mRNA and protein upregulation preceded the radiation-induced pulmonary fibrosis, suggesting a pathogenetic role in the development of radiation fibrosis.

The intratracheal administration of endotoxin: X. Dexamethasone downregulates neutrophil emigration and cytokine expression in vivo.

Intratracheal instillation of endotoxin (LPS) causes acute pulmonary inflammation characterized by the accumulation of plasma proteins and leukocytes within the pulmonary airways. The synthetic glucocorticoid dexamethasone 1) inhibits the LPS-initiated vascular leak of plasma proteins into the airspace, 2) inhibits the LPS-initiated emigration of neutrophils and lymphocytes into the airspace in a dose-dependent fashion, and 3) inhibits LPS-initiated mRNA and/or bronchoalveolar lavage protein expression of cytokines (TNF, IL-1 and IL-6) and chemokines (MIP-1 alpha, MIP-2 and MCP-1). In conclusion, dexamethasone inhibits both the vascular and cellular aspects of acute inflammation by downregulation of a broad spectrum of inflammatory cytokines and chemokines.

Keratinocyte growth factor decreases pulmonary edema, transforming growth factor-beta and platelet-derived growth factor-BB expression, and alveolar type II cell loss in bleomycin-induced lung injury.

Keratinocyte growth factor (KGF), a potent growth factor for type II pneumocytes and Clara cells, has been shown to prevent the end-stage pulmonary fibrosis and mortality in a rat model of bleomycin-induced lung injury. In this study, protective effects of KGF were explored during the earlier course of bleomycin-induced lung injury by studying protein exudation in alveolar edema fluids, pulmonary expression of transforming growth factor-beta (TGF beta) and platelet-derived growth factor-BB (PDGF-BB), and changes in type II pneumocytes and Clara cells after i.t. (intratracheal) bleomycin injection following KGF- or saline-pretreatment in rats. Total protein in bronchoalveolar lavage (BAL) fluids after bleomycin injury from KGF-pretreated rats was significantly lower than the levels in saline-pretreated rats. TGF beta protein in BAL fluids which peaked at day 3 after i.t. bleomycin in saline-pretreated lungs was not significantly increased at any time points in KGF-pretreated rats. PDGF-BB protein in whole lung tissues of KGF-pretreated rats also remained near normal throughout the course after i.t. bleomycin, in contrast to the significant increase in saline-pretreated rats. Numbers of type II pneumocytes and Clara cells in KGF-pretreated lungs after a high dose of bleomycin were close to the normal in intact lungs. At the same dose of bleomycin injury, type II pneumocytes in saline-pretreated lungs were markedly decreased, while the number of Clara cells in these rats was relatively preserved as the pre-injury level. In conclusion, KGF prevents bleomycin-induced end-stage pulmonary injury and mortality probably at least partly by decreasing protein-rich pulmonary edema, protein expression of fibrogenic cytokines TGF beta and PDGF-BB, and type II cell loss during the course of lung injury.

Intratracheal administration of endotoxin and cytokines: VIII. LPS induces E-selectin expression; anti-E-selectin and soluble E-selectin inhibit acute inflammation.

E-selectin is an inducible endothelial adhesion molecule that binds neutrophils. E-selectin mRNA is not constitutively detectable in the lungs of rats. Intratracheal injection of LPS induces pulmonary E-selectin mRNA expression at 2-4 h. Intratracheal injection of LPS followed at 2 and 4 h by intravenous injection of mouse F(ab')2 or F(ab') anti-E-selectin monoclonal antibody inhibits the emigration of neutrophils into the bronchoalveolar space at 6 h by 50-70%. TNF and IL-6 bioactivity are not decreased in bronchoalveolar lavage fluid after treatment with anti-E-selectin antibody as compared to controls, suggesting that the anti-E-selectin does not affect the magnitude of the LPS-initiated cytokine cascade. Intratracheal injection of LPS followed at 2 and 4 h by intravenous injection of soluble E-selectin inhibits neutrophilic emigration at 6 h by 64%, suggesting that endogenous soluble E-selectin shed from activated endothelium may play a role in the endogenous down-regulation of acute inflammation. E-selectin-mediated adhesion of neutrophils to endothelium appears crucial to the full development of the acute inflammation response.