Characterization of M1/M2 Tumour-Associated Macrophages (TAMs) and Th1/Th2 Cytokine Profiles in Patients with NSCLC.

Lung cancer is one of the most commonly reported cancers, and is known to be associated with a poor prognosis. The function of tumour-associated macrophages (TAMs) in lung cancer patients is multifaceted and the literature shows conflicting roles. (I) To analyze the Th1 and Th2 cytokine levels that contribute to the differentiation of M1 and M2 macrophage populations in the serum of patients with NSCLC versus non-cancer controls; and (II) To characterize the M1 and M2 macrophage populations within TAMs in different subtypes of NSCLC compared to non-tumour tissue. The Th1 and Th2 cytokine levels were analyzed in serum using the Bio-Plex assay. In addition, TAMs subsets from non-tumour and tumour tissues were analyzed using immunohistochemistry (IHC). The level of IL-1ß, IL-4, IL-6 and IL-8 was found to be increased in the serum of patients with large cell carcinoma but not in other NSCLC subtypes compared to non-cancer controls. In addition, the expression of CD68 and M2 marker CD163 was found to be increased (P ≤ 0.0001) in all NSCLC subtypes compared to non-tumour tissues. In contrast, the expression of iNOS (M1 marker) was decreased in the tumour tissue of patients with adenocarcinoma (P ≤ 0.01) and squamous carcinoma (P ≤ 0.05) but not in large cell carcinoma compared to non-tumour tissue. The results of this study indicate that NSCLC might have the ability to alter phenotype within the lung tumour areas in the local environment (TAMs) but not in the bloodstream in the systemic environment (serum) except for large cell carcinoma.

Intracellular cyclic nucleotide analogues inhibit in vitro mitogenesis and activation of fibroblasts derived from obstructed rat kidneys.

As several studies indirectly suggest that inhibiting the intracellular breakdown of cyclic nucleotides may inhibit fibrogenesis, this study used membrane permeable cyclic nucleotide analogues to examine the role of cAMP and cGMP signaling pathways in the regulation of renal fibroblast function. Fibroblasts were isolated by explant outgrowth culture of rat kidneys post unilateral ureteric obstruction. Subcultured cells were exposed to 10- 1,000 microM of the cyclic nucleotide analogues 8-bromo-cAMP (8br-cAMP) and 8-bromo-cGMP (8br-cGMP). Functional parameters examined included mitogenesis (thymidine incorporation), collagen synthesis (proline incorporation), myofibroblast differentiation (Western blotting for alpha-smooth muscle actin; alpha-SMA) and expression of CTGF (Northern blotting), a TGF-beta(1)-driven immediate early response gene. Serum-stimulated mitogenesis was decreased 27 +/- 4% by 100 microM 8br-cAMP (p < 0.01), 49 +/- 6% by 1,000 microM 8br-cAMP (p < 0.001) and 43 +/- 7% by 1,000 microM 8br-cGMP (p < 0.01). 1,000 microM 8br-cAMP and 8br-cGMP reduced basal collagen synthesis by 80 +/- 5 and 60 +/- 21% respectively (both p < 0.05). Maximum dose of 8br-cAMP but not 8br-cGMP inhibited basal expression of the differentiation marker alpha-SMA by 43 +/- 33 (p < 0.05), resulted in a more rounded cell morphology and reduced expression of CTGF by 39 +/- 24% (p < 0.05). Measurement of mitochondrial activity confirmed that effects were independent of cell toxicity. In conclusion, cyclic nucleotides inhibit fibrogenesis in vitro. Strategies which elevate intracellular cyclic nucleotide concentrations may therefore be therapeutically valuable in preventing the proliferation and activation of fibroblasts in progressive renal disease.

Sox18 is transiently expressed during angiogenesis in granulation tissue of skin wounds with an identical expression pattern to Flk-1 mRNA.

Sox18 encodes a member of the Sry-related high mobility group box (SOX) family of developmental transcription factors. Examination of Sox18 expression during embryogenesis has shown that Sox18 is expressed transiently in endothelial cells of developing blood vessels, and mutations in Sox18 have been found to underlie the mouse vascular and hair follicle mutant ragged. In this study we have examined the expression of Sox18 in angiogenesis during wound healing. Full-thickness skin wounds were created in mice, and subsequent expression of vascular endothelial growth factor (VEGF), the VEGF receptor Flk-1, alpha1 (iv) collagen (Col4a1), and Sox18 were studied using in situ hybridization. As has been previously reported, VEGF was expressed predominantly in the keratinocytes at the wound margins. Sox18 expression was found five days after wounding during capillary sprouting in granulation tissue and persisted through the proliferative phase of healing, but was not detected in fully epithelialized wounds 21 days after wounding. Sox18 mRNA expression was detected in capillaries within the granulation tissue and showed an identical pattern of distribution to Flk-1 and Col4a1 mRNA expression in endothelial cells. Immunostaining with a polyclonal anti-Sox18 antibody showed SOX18 protein localized in capillary endothelial cells within the granulation tissue. Capillaries in the subcutaneous tissue of unwounded skin showed no Sox18 expression. Sox18 may therefore represent a transcription factor involved in the induction of angiogenesis during wound healing and tissue repair, but not in the maintenance of endothelial cells in undamaged tissue.

Human renal fibroblast contraction of collagen I lattices is an integrin-mediated process.

BACKGROUND: Expression of the beta1 family of integrins allows dermal fibroblasts in wounds to contribute to the healing process through migration, adhesion, synthesis, and rearrangement of extracellular matrix. To date the ability of human renal fibroblasts to reorganize collagens and the role of cell surface receptors in this process remain unknown. METHODS: Renal fibroblasts were grown from the cortical tissue of surgically removed human kidneys. The ability of human renal fibroblasts to reorganize interstitial collagen I was examined in vitro using solidified collagen I lattices. Integrin function was blocked by incubating fibroblasts with isotype-specific antibodies prior to addition to collagen I lattices. RESULTS: Human renal fibroblasts embedded in collagen I lattices progressively decreased lattice diameter to 60.6+/-11.4% of initial diameter at 48 h post-release (P:<0.01). Fibroblasts incubated in the presence of antibody to beta1 integrin failed to contract collagen I lattices, whilst fibroblasts incubated with non-specific antibody reduced lattice diameter to 60.1+/-12.4% of initial diameter at 48 h post-release (P:<0.01). Further characterization of integrin alpha subunits showed that blocking alpha2beta1 integrin prevented lattice contraction (P:<0.05, alpha2beta1 integrin antibody vs non-specific antibody), whilst blocking of alpha5beta1, alpha3beta1 and alpha1beta1 integrins did not influence this process. CONCLUSIONS: We postulate that collagen I fibril rearrangement by human renal fibroblasts in vitro appears to be an integrin-mediated process involving the alpha2beta1 integrin.

Cautionary note on the use of end-labelling DNA fragments for detection of apoptosis.

The gastrointestinal epithelum undergoes a continual process of cell renewal which is partly regulated by apoptosis. The process of growth and differentiation is greatly enhanced in the terminal ileum after massive small bowel resection, a well-established model of intestinal adaptation. We have applied the terminal deoxynucleotidyl transferase (TdT)-mediated UTP nick end-labelling (TUNEL) method to establish the role of apoptosis in intestinal adaption in terminal ileum of control animals after small bowel resection. Healing skin wound and lymph node were used as positive control tissue for apoptotic cells. We report that considerable inter-animal variation was observed in the intestinal tissue. We believe that caution is required in the interpretation of TUNEL staining in intestinal tissue and in its use as a specific marker for apoptosis in this setting.

Evolution of tubulointerstitial fibrosis in experimental renal infection and scarring.

Renal tubulointerstitial fibrosis may result from a loss of tubulointerstitial volume, which produces a disproportionate increase in the density of matrix. This study examines the relationship between fibrogenesis and collapse in scar formation after experimental renal infection. Escherichia coli were inoculated into the renal cortex of Sprague Dawley rats, with saline substituted in a control group. Glomerular, tubular, and interstitial profile areas were determined. Density of glomerular profiles was used as a measure of tubulointerstitial collapse. Collagen type I, III, and IV expression was examined by in situ hybridization and immunohistochemistry. Myofibroblasts were identified by alpha smooth muscle actin immunohistochemistry, and matrix metalloproteinase-1 (MMP-1) and MMP-2 were localized with appropriate antisera. Acute interstitial edema was followed by increasing density of glomerular profiles, paralleled by loss of interstitial volume and progressive tubular atrophy. Glomerular profile area remained unchanged. Density of glomerular profiles was not temporally related to myofibroblast accumulation. Procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) transcription was focal, spatially related but temporally ordered. Collagen I, III, and IV immunostaining was increased from days 3, 24, and 100, respectively (P < 0.05 versus day 0 and day 100 saline). However, when corrected for glomerular density, collagen I immunostaining decreased between days 24 and 100, whereas collagen III and IV no longer differed from day 0. MMP staining within the lesion was confined to occasional interstitial and epithelial cells throughout. It is concluded that in this model, contraction and collapse of the tubulointerstitial parenchyma has a greater influence than new collagen production on final fibrotic density.

Amylin as a growth factor during fetal and postnatal development of the rat kidney.

We have previously reported that amylin has mitogenic actions on tubular epithelial cells isolated from mature rat kidney and cultured in vitro. In experiments using in situ hybridization, we have demonstrated that amylin mRNA can be detected transiently in rat metanephros from embryo day 17 (E17) to postnatal day 3 (PN3). These transcripts are localized in the sub-nephrogenic zone. RT-PCR was performed using oligonucleotide primers for rat amylin and mRNA extracted from fetal body (E19), PN1 and PN5 metanephroi, and adult rat kidney. These results corroborate the finding, using in situ hybridization, that there is a window of expression of rat amylin in the developing kidney in the perinatal period. During this period tubular elongation is evident and amylin peptide, detected by immunohistochemical staining, is found associated with developing tubules. Some of these tubules also express a brush border glycoprotein, detected by immunohistochemical staining. Amylin acts as a mitogen with primary cultures of proximal tubular epithelial cells from PN4 renal cortex. An amylin antagonist inhibited this mitogenic action suggesting that this was mediated by amylin receptors as previously described. We suggest that amylin peptide is biosynthesized in the developing proximal tubules, acts in an autocrine fashion to promote the proliferation and differentiation of brush border epithelial cells and hence plays an important role as a growth factor in the development of the kidney.

Increased density of renal amylin binding sites in experimental hypertension.

High-affinity binding sites for the pancreatic beta-cell hormone amylin have been reported in the kidney, and it has been postulated that these sites may be involved in the genesis of hypertension. In the present study, we have used in vivo injection of 125I-amylin and in vitro autoradiographic techniques to assess renal amylin binding in both a genetic and a surgically induced model of hypertension. In the spontaneously hypertensive rat (SHR) at 6 weeks of age, before the rise in systolic blood pressure, there was a 36% increase in density of amylin binding compared with their normotensive counterpart, the Wistar-Kyoto rat (WKY). In SHR, there was a further increase in the density of amylin binding (to 53% greater) as the systolic blood pressure rose between 6 and 12 weeks of age. Histological examination of kidneys from SHR at 12 weeks of age revealed staining for a brush border glycoprotein, normally restricted to the proximal tubules, extending from the urinary pole into half of the epithelial lining of the glomerular capsule. In contrast to WKY, these cells also bound 125I-amylin with high density in SHR. In a rat model of renal ablation and hypertension, systolic blood pressure correlated with the density of 125I-amylin binding in the renal cortex (r=.54, P=.003, n=28). The changes in amylin binding reported here suggest a possible role for this peptide and/or activation of its receptor in the genesis as well as the maintenance of hypertension.

Apoptosis is increased in a model of diabetes-impaired wound healing in genetically diabetic mice.

Impaired wound healing is a common complication of diabetes mellitus. The underlying pathophysiology of diabetes-impaired healing is poorly understood. In the present study we have compared cell proliferation rates, apoptosis (programmed cell death), the myofibroblast marker alpha-smooth muscle actin and procollagen I mRNA expression, between diabetic and control mice. Full-thickness skin wounds were made in non-obese diabetic (NOD) mice and C57B6 controls. NOD mice showed a marked retardation of wound healing at both 7 and 14 days after wounding. Comparison of cell proliferation rates 7 days after wounding, using 5-bromo-2'-deoxy-Uridine incorporation, showed higher rates of cell proliferation in controls (88.1 +/- 12.8) than in NOD wounds (52.1 +/- 9.9, p < 0.02, n = 4). Immunohistochemical detection of alpha-smooth muscle actin, showed a later onset in diabetic wounds, suggesting that wound contraction may be delayed in the diabetic animals. In situ hybridisation for alpha 1 (I) procollagen mRNA expression, showed reduced procollagen I expression in the diabetic wounds when compared with controls. Lastly, there appeared to be higher levels of apoptosis in diabetic wounds, shown by the terminal transferase mediated UTP nick end-labelling technique. Apoptotic cells were rare in control wounds confirming previous studies, which showed that apoptosis occurs late in normal wound healing as the wound matures into scar tissue. In conclusion, we hypothesize that reduced cell proliferation, retarded onset of the myofibroblast phenotype, reduced procollagen I mRNA expression and aberrant control of apoptotic cell death may contribute to impaired wound healing seen in this diabetic model.

In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys.

Recent evidence suggests that a local renin-angiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin II formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the light-microscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial cells and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day E18 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohistochemistry showed staining of relatively few electron-dense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.

Vascular expression of clusterin in experimental cyclosporine nephrotoxicity.

Clusterin is a widely expressed glycoprotein, which appears to be induced during tissue damage. We have examined the expression of the clusterin gene in the kidney during the development of cyclosporine (CyA)-induced nephrotoxicity in the rat using in situ hybridization histochemistry. Female Sprague-Dawley rats (170 g) were divided into experimental or control groups and were given intraperitoneal injections of CyA (25 mg/kg) or vehicle respectively for 2, 4 and 6 weeks. Kidneys from animals sacrificed at these times were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were hybridized using either a sense or antisense riboprobe complementary to clusterin mRNA which was labelled with 32P-UTP. Clusterin gene expression was detected in scattered tubules in kidneys from control animals. Expression of clusterin in cortical collecting ducts of CyA-treated animals was evident at 2 weeks and increased substantially at 4 and 6 weeks. Clusterin expression was also seen in afferent arterioles, the glomerular capsule and transitional epithelium of the renal pelvis of kidney sections from rats treated with CyA for 6 weeks. No labelling above background was seen at any time with the sense probe. Renin immunostaining in afferent arterioles of kidney sections from animals treated with CyA showed a marked increase after 4 and 6 weeks of CyA treatment.

Erythropoietin gene expression in fetal and adult sheep kidney.

The exact location of the cells which express erythropoietin (Epo) in the kidney is still controversial, with conflicting reports suggesting that both peritubular interstitial cells or proximal tubular epithelial cells are possible sites of Epo expression. In the present study we have examined the location of Epo-expressing cells in fetal and adult sheep kidneys after Epo expression was stimulated by anaemia. In situ hybridization histochemistry was performed using synthetic oligonucleotide probes complementary to part of the sheep Epo cDNA sequence or a riboprobe (cRNA) for ovine Epo of 520 bases. Epo expression was confined to peritubular cells of the kidney cortex, in general in the area close to the cortico-medullary junction. In some severely anaemic adult sheep kidneys, Epo-expressing cells were also found in the outer cortex. In addition we located Epo-expressing cells in the kidneys not only of anaemic fetuses (89-140 d of gestation, term = 150d) but also in kidneys from normal fetuses 60-110d of gestation. Again, Epo expression was seen only in peritubular cells of the kidney cortex. These findings confirm that the kidney is an important site of Epo production, in the sheep, from at least 0.4 gestation, but also show that there is no ontogenic change in the cellular site of production within the kidney.

Cellular and ultrastructural location of angiotensinogen in rat and sheep kidney.

Recent evidence suggests the involvement of a local renin-angiotensin system in some renal actions of angiotensin II (Ang II). In this study the renal distribution of the precursor to angiotensin formation, angiotensinogen, was investigated in rats and sheep using immunohistochemistry, immunoelectron microscopy and non-isotopic hybridization histochemistry. Immunostaining for angiotensinogen was seen in proximal tubules (PCT) of both rat and sheep kidneys. In the rat the strongest immunostaining was found in the kidneys of neonatal (1 day old) rats. Staining declined after birth. Non-isotopic hybridization histochemistry using oligodeoxynucleotide probes labeled with biotin confirmed the presence of angiotensinogen mRNA expression in PCT of the rat renal cortex. Electron microscopic immunohistochemistry using antibodies raised against rat angiotensinogen showed weak staining in the adult of granule-like structures close to the apical membrane of PCT cells. In the neonatal rat kidney, angiotensinogen immunostaining was found throughout the PCT cells and was markedly stronger than that seen in adult rat kidney. In sheep, angiotensinogen immunostaining with an antibody raised against purified ovine angiotensinogen showed staining of PCT in fetal, newborn and adult sheep kidney. The strongest immunostaining seen was in fetal sheep kidney with a decline seen after birth. Reverse transcription polymerase chain reaction (RT-PCR) showed that angiotensinogen mRNA was expressed in the sheep kidney at all ages studied. Angiotensinogen expression was higher in fetal sheep kidneys (77 day and 141 day gestation) than in adult sheep kidney. In conclusion, angiotensinogen mRNA expression was detected in both rat and sheep kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal gene expression.

The advent of cloned DNA probes has revolutionized the study of cells and tissues via the technique of hybridization histochemistry. Because of the intrinsic specificity of the probes the application of the technique to functionally complex and morphologically diverse tissue like the kidney has been especially rewarding. The technology has been advanced enormously by the parallel development of synthesized DNA or oligonucleotides. A specific synthetic probe can be manufactured in a day, purified, labeled and used to determine in which cells a particular gene of interest is being expressed ("switched on"). The applications of this technology are particularly opposite in the renal field. They can provide unique insights into normal physiological processes and will provide new diagnostic approaches of unparalleled specificity. Some specific examples have been chosen over a wide range of institute programs to highlight the potential value of the technique of hybridization histochemistry and to emphasize its potential for studying renal physiology and pathology.

Thyroid hormone receptors and stimulation of angiotensinogen production in HepG2 cells.

Binding characteristics and effects of 3,5,-3'-triiodo-L-thyronine (T3) on angiotensinogen production in HepG2 were studied in serum-free medium. Binding was performed on intact cells and on partially purified isolated nuclei using [125I]T3. Scatchard plots revealed one class of high affinity binding sites with a Kd of approximately 80 pmol/liter. Calculation of maximum binding showed that HepG2 possess approximately 1000 binding sites per cell. Unlabeled T3 and T4 competed for binding sites on intact HepG2 with 50% inhibition of [125I] T3 binding at approximately 3.0 and 38.0 pmol/liter, respectively. The HepG2 showed a dose-dependent increase in angiotensinogen production in serum-free medium which was maximal at 10(-5) mol/liter (two-fold increase/10(6) cells/24 h) and had an EC50 of approximately 5.0 x 10(-8) mol/liter. T3 also produced after 24 h a dose-dependent increase in DNA highly correlated with T3 applied (r = 0.88, P less than 0.01). In conclusion, this study shows that HepG2 possess specific high affinity binding sites for T3 and that T3 stimulates angiotensinogen production and DNA synthesis in these cells.

Regulation of angiotensinogen production by angiotensin II analogues.

Specific binding sites for angiotensin II (Ang II) were identified in a human hepatoma cell line, HepG2. Binding of [125I]-Sar1 Ang II to these cells showed a high-affinity site with a Kd of 2.4 +/- 0.2 nmol/l. This specific binding was not changed during the cell cycle and showed no alteration after 24 h of treatment with Sar1-Ang II (10(-8) mol/l). Exposure of HepG2 cells to the Ang II agonist Sar1-Ang II caused a dose-dependent decrease in angiotensinogen production. The maximal inhibitory effect was at a dose of 10(-6) mol/l Sar1-Ang II which elicited 67% inhibition of angiotensinogen production after 24 h (control: 2.015 +/- 0.5 micrograms angiotensinogen/mg DNA; Sar1-Ang II 10(-6) mol/l: 0.68 +/- 0.03 micrograms angiotensinogen/mg DNA). Fifty per cent inhibition was obtained at a dose of 10(-9) mol/l Sar1-Ang II. Angiotensin II had a less marked effect, showing maximal inhibition of 40%. This study shows that the HepG2 cells possess specific Ang II binding sites and that Ang II analogues induce a dose-dependent inhibition of angiotensinogen production in cell culture.

Hybridization histochemistry.

The location of gene expression by hybridization histochemistry is being applied in many areas of research and diagnosis. The aim of this technique is to detect specific mRNA in cells and tissues by hybridization with a complementary DNA or RNA probe. Requirements for optimal specificity, sensitivity, resolution and speed of detection may not all be encompassed in one simple technique suitable for all applications, thus appropriate procedures should be selected for specific objectives. With reference to published procedures and our own extensive experience, we have evaluated fixatives, probes, labels and other aspects of the technique critical to the preservation and hybridization in situ of mRNA and detection and quantitation of hybrids.