Stromal Macrophages Research Articles

Central Role of Interleukin-15 in Postovulatory Recruitment of Peripheral Blood CD16(−) Natural Killer Cells into Human Endometrium

A large number of CD16(-) natural killer (NK) cells appear in the human endometrium after ovulation. One possible explanation for this phenomenon is recruitment from peripheral blood (PB) CD16(-) NK cells. In this study, we examined whether IL-15 is involved in postovulatory recruitment from PB CD16(-) NK cells. The IL-15 receptor alpha chain was expressed on PB CD16(-) NK cells but not on CD16(+) NK cells. In an in vitro migration assay, recombinant human IL-15 enriched PB CD16(-) NK cells. Endometrial soluble protein fraction in the secretory phase, but not in the proliferative phase, also enriched these NK cells. Neutralization of IL-15 in the secretory phase endometrium with anti-IL-15 monoclonal antibody significantly lowered the enrichment of PB CD16(-) NK cells. In contrast, neutralization of other potential chemokines, including stromal cell-derived factor-1 or macrophage inflammatory protein-1alpha, had no significant effect. The IL-15 concentration in the endometrial soluble protein fraction was higher in the secretory phase than in the proliferative phase, with a peak in the midsecretory phase. These results support the idea that endogenous IL-15 in secretory phase endometrium plays a central role in postovulatory recruitment of PB CD16(-) NK cells into the human endometrium.

The Fibroblastic Coconspirator in Cancer Progression

A remarkable change has occurred in the thinking about epithelial-derived cancer in recent years: From almost entirely focusing on oncogenes and tumor suppressor genes has come the realization that the tumor microenvironment is a coconspirator in the carcinogenic process. Many types of stromal cells, including fibroblasts, adipocytes, macrophages, mast cells, and cells of the vascular system, are crucial contributors to epithelial carcinogenesis. Here, we focus on the fibroblast's role in cancer progression and the molecules involved in the communications between the fibroblasts and the cancer cells, including fibroblast secreted protein 1 (FSP-1 or S100A4), transforming growth factor beta (TGF-beta), the chemokine CXCL-12 (stromal derived factor 1 alpha, SDF-1alpha), type I collagen, and matrix metalloproteinase 13 (MMP-13).

Stimulation of the Supportive Function of Marrow Stromal Cells by Anticancer Agents: Critical Role of Marrow Macrophages.

Anticancer agents used for the treatment of hematological malignancies are known to cause long-term toxicities to the structure and function of bone marrow microenvironment. These obbservations are based predominantly on the in vivo experiments with anticancer drug-treated mice and also on the clinical features of the patients who survived after inhtensive chemotherapies. In the present study, in order to evaluate immediate gene response of human marrow stromal cells to anticancer drugs, we tested with real-time PCR for the expression of various cytokine mRNAs from the stromal cells treated with anticancer drugs. Contrary to our expectation, we found that treatment of stromal cells with cytarabine (Ara-C; 0.1–100 μmol) for 3 to 14 days significantly and dose-dependently enhances the expression of mRNAs of stem cell factor (SCF) and leukemia inhibitory factor (LIF) while downregulates interleukin-6 (IL-6) mRNA. On the other hand, carboplatin (0.1–100 μmol) up-regulated only SCF mRNA and adriamycin (0.001–1 μmol) did not affect these gene expressions (n=6). In order to determine the responsive cell elements of stromal cells for these novel responses to Ara-C, mRNAs were independently quantified after separating CD14 positive macrophages and CD45 negative mesenchymal cells from Ara-C-treated stromal cells (n=6). In this additional experiments, we found that the above-mentioned changes in gene expression by Ara-C were provoked predominantly in the stromal macrophage fraction. Based on these observations, we treated the stromal cells established from 6 patients with AML and 11 with non-leukemic subjects with Ara-C for 2 weeks, followed by washing 3-times to eliminate Ara-C. Allogenic CD34 positive cells were then recharged and supportive functions of the stromal cells were evaluated with standard 2-stage long-term cultures. Indeed, the results showed that transient treatment with Ara-C significantly and dose-dependently upregulates supporting function of premature hematopoietic cells in non-leukemic and, although to the less extent, in leukemic stromal cells. These novel short-term stimulatory effects of Ara-C to marrow microenvironment may provide new explanations for some clinical events such as mechanism of rapid recovery of normal hematopoiesis and stem cell mobilization observed several days after completion of intensive chemotherapy to acute leukemia. In addfition, the pattern of gene response of stromal cells induced by Ara-C is of biologically great interest since it is quite different from the already known gene response of human stromal cells provoked by IL-1.

Specific Enhanced Expression of Platelet-Derived Endothelial Cell Growth Factor in Submucosa of Human Colorectal Cancer

Platelet-derived endothelial cell growth factor, identified to be an angiogenic factor, has been implicated in metastases of colorectal cancer. This study aimed to clarify the role and localization of platelet-derived endothelial cell growth factor associated with human colorectal cancer invasion. Thirty-two patients with colorectal cancer who had undergone surgery were analyzed. Platelet-derived endothelial cell growth factor enzyme activities in the colorectal cancer specimens were measured. Cells that expressed platelet-derived endothelial cell growth factor were identified and localized by immunohistochemical analysis with anti-human platelet-derived endothelial cell growth factor antibody and by in situ hybridization with specific RNA probe. Platelet-derived endothelial cell growth factor enzyme activity increased significantly in cancer tissues compared with normal colonic mucosa at various distances from the cancer. Immunohistochemical analysis and in situ hybridization demonstrated platelet-derived endothelial cell growth factor expression in stromal macrophages and fibroblasts located in cancer tissues and surrounding noncancerous tissues, although the tumor cells and normal colonic mucosa were negative. The value of platelet-derived endothelial cell growth factor expression was highest at the border of the colorectal cancer (35.3 +/- 8.9 percent), followed by the cancer nest (15.2 +/- 9.2 percent) and normal mucosa (7.7 +/- 3.4 percent). In the border area, the highest value of platelet-derived endothelial cell growth factor expression was observed in the submucosa (35.3 +/- 8.9 percent), followed by the muscular propria (21.9 +/- 7.7 percent) and the subserosa (14.9 +/- 5.5 percent). Stromal macrophages and fibroblasts are responsible for elevated platelet-derived endothelial cell growth factor activity in colorectal cancer. The significance of enhanced expression of platelet-derived endothelial cell growth factor in the submucosa at the cancer border remains unclear. Cancer stroma may be an important factor for cancer angiogenesis and may serve as a treatment target through specific modulation of angiogenic factors.

Interleukin-8 is involved in cervical dilatation but not in prelabour cervical ripening.

Our aim was to determine the amount and source of interleukin (IL)-8 and to study IL-8 receptor expression in the human cervix during pregnancy and after labour. Cervical biopsies were obtained from six non-pregnant women, eight women undergoing pregnancy termination, 17 women undergoing elective caesarean section and 11 women after vaginal delivery. IL-8 levels were compared in women with and without a ripe cervix, as determined by cervical Bishop score and cervicovaginal fetal fibronectin levels. Levels of IL-8 and IL-1beta, a regulator of IL-8 expression, were determined by enzyme-linked immunosorbent assay (ELISA). IL-8, IL-1beta and IL-8 receptor proteins were localized by immunohistochemistry. Compared with late pregnancy, IL-8 levels increased after labour and vaginal delivery (P < 0.01) but there was no correlation with cervical ripening. IL-8 was localized to stromal cells, macrophages and granulocytes. There were no significant differences in IL-1beta levels between groups. IL-8 receptors were expressed primarily on granulocytes and macrophages after vaginal delivery. We conclude that IL-8 is involved in cervical dilatation but not in cervical ripening.

Clinical significance of VEGF-C status in tumour cells and stromal macrophages in non-small cell lung cancer patients.

Recent experimental studies have revealed that tumour-associated stromal macrophages as well as tumour cells express vascular endothelial growth factor C (VEGF-C), which plays important roles in lymphangiogenesis, which is a critical factor in the progression of many malignant tumours including non-small-cell lung cancer (NSCLC). However, no clinical study on VEGF-C expression in both stromal macrophages and tumour cells has been reported, and we conducted the present study to address the issue in resected NSCLC. A total of 206 patients with completely resected pathologic stage I–IIIA NSCLC were retrospectively reviewed. Expression of VEGF-C in primary lung tumour was assessed immunohistochemically. Expression of VEGF-C in tumour cells was high in 125 patients (60.7%), and that in stromal macrophages was positive in 136 patients (71.2%). The status of VEGF-C in tumour cells or in stromal macrophages was not correlated with nodal status or angiogenesis. The 5-year survival rate of high tumoral VEGF-C patients (60.7%) was significantly lower than that of low tumoral VEGF-C patients (39.3%) (P=0.046), and a multivariate analysis confirmed that tumoral VEGF-C status was a significant and independent prognostic factor. Moreover, tumour showing high VEGF-A and VEGF-C expression in tumour cells showed the poorest prognosis (5-year survival rate, 45.1%). The status of VEGF-C in stromal macrophages was not correlated with the prognosis. In conclusion, tumoral VEGF-C status, not stromal VEGF-C status, was a significant prognostic factor in resected NSCLC.

Induction of adipogenesis by the intrasplenic transplantation of chick serum clots.

Chick serum contains a factor that stimulates adipogenesis in Meckel's chondrocytes in vitro. The present study examined whether chick serum has a capacity for adipogenic induction in vivo, by transplanting serum clots (created by drying chick serum for up to 4 weeks) into mouse spleens. Specimens were harvested for histological analyses, which included light and electron microscopy and immunohistochemistry. The transplanted serum clots induced the appearance of lipid droplet-containing cells in splenic cords and sinus. Almost all the lipid droplet-containing cells were positive for sudan staining and consisted of multilocular lipid vacuoles. Immunostaining showed that the adipocytes induced by transplantation of the serum clots initially appeared as peroxisome proliferator-activated receptor-gamma (PPARgamma)-positive cells and developed into leptin and alpha-glycerophosphate dehydrogenase (GPDH)-producing cells, in addition to type III collagen synthesis. Furthermore, double immunofluorescence staining revealed that the immunoreactivity for GPDH was detected not only in stromal cells but also in macrophages. It was thus confirmed that stromal cells and macrophages in the spleen contain lipid droplets as seen in intact white adipose cells. The present results suggest that chick serum contains factors for adipocyte induction not only in vitro but also in vivo, and that the adipogenic potential does not depend on the supplements used during the cell culture.

Platelet-derived endothelial cell growth factor expression is an independent prognostic factor in colorectal cancer patients after curative surgery

Aims. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor that undergoes increased expression in colorectal carcinomas, but its prognostic value is a topic of debate. The aim of this study is to clarify the prognostic value of PD-ECGF expression in colorectal carcinomas. Methods. PD-ECGF expression was measured by enzyme-linked immunosorbent assay in frozen materials from 134 colorectal cancer patients who had recived curative resections. Patients were divided into high expression and low expression groups based upon selected cut-off value. Correlations among PD-ECGF expression, clinicopathologic features, and disease-free interval were studied by univariate and multivariate analysis. To evaluate the origin of PD-ECGF, serial sections of the 134 tumours were stained for PD-ECGF and CD68. Results. PD-ECGF expression in the normal mucosa was 34.4±15.5 (Units/mg protein) and the cut-off value was 65.4 (mean+2SD). There were no significant correlations between clinicopathological features and PD-ECGF expression. The disease-free interval for the high PD-ECGF expression group was significantly longer than that of the low expression group ( P=0.05). A multivariate Cox's regression analysis revealed that high PD-ECGF expression is an independent factor for better outcome. In immunohistochemical study, almost all tumour cells were negative for PD-ECGF, but stromal macrophages were predominantly positive for PD-ECGF. Conclusions. The PD-ECGF expression originated from stromal macrophages was a predictor for favorable outcome after curative resections for colorectal cancer.

Laminin-5 gamma 2 chain is colocalized with gelatinase-A (MMP-2) and collagenase-3 (MMP-13) in odontogenic keratocysts.

Odontogenic keratocyst (KC) differs from other epithelial odontogenic cysts in regard to increased epithelial proliferation and a strong tendency to recur. Laminin-5 (Ln-5) is an epithelial anchoring filament component, which after modulation by certain matrix metalloproteinases (MMPs), like MMP-2 and MMP-13, induces epithelial cell migration. Using in situ hybridization and immunohistochemistry, we studied the Ln-5 gamma-2 chain expression related to the expression of MMP-2, -8, and -13 in different odontogenic cysts, including radicular cysts (RC; n = 11), follicular cysts (FC; n = 11), and odontogenic keratocysts (KC; n = 16). Ln-5 mRNA was present in all cysts examined, while less than half of KCs and RCs (33 and 40%, respectively) demonstrated MMP-2 mRNA. MMP-13 mRNA was present in all KC samples. Ln-5 protein was located as a continuous ribbon in BM zone of all KCs, and MMP-2 and MMP-13 immunoreactivities colocated significantly with Ln-5 in that area. MMP-8 was expressed by stromal macrophages and epithelial goblet cells, but never located in BM zone. Our results indicate that the colocalization of Ln-5 with MMP-2 or MMP-13, but not with MMP-8, in BM zone of KCs, may be related to special characteristics of KC.

Tissue factor-dependent coagulation activation and impaired fibrinolysis in situ in gastric cancer.

Thromboembolism frequently complicates gastric cancer. This study examined the solid phase interaction between gastric cancer and coagulation proteins in situ that may explain coagulation activation and that may contribute to tumor progression and angiogenesis in this tumor type. Immunohistochemical techniques were applied to tissues from 37 cases of adenocarcinoma of the stomach obtained at surgical resection. Fibrinogen was present throughout the tumor stroma. Fibrin and its D-dimer cross-link sites occurred at the host-tumor interface. Subunit "a" of factor (F) XIII and F VII, IX, X, and XII were observed on cancer cells. Prothrombin and prothrombin fragment F1+2 (F1+2) were demonstrated in the tumor stroma on cancer cells and on small blood vessels. Tissue factor (TF) was present on cancer cells and tumor-associated macrophages. Protein C was observed on cancer cells and small blood vessels, whereas protein S was present only in the vascular bed. There was no staining for tissue factor pathway inhibitor (TFPI). High-molecular-weight (HMW) urokinase plasminogen activator (u-PA) antigen was not detected, but weak and inconsistent staining for low-molecular-weight (LMW) u-PA was demonstrated on cancer cells. Weak staining for tissue plasminogen activator (t-PA) occurred on cancer cells and in the tumor stroma. In contrast, plasminogen activator inhibitor-1 (PAI-1) expression was strong in the tumor stroma, along with PAI-2 and PAI-3. The endothelium of small stromal blood vessels, particularly near the host-tumor interface, demonstrated von Willebrand factor antigen (vWF Ag). Vascular endothelial growth factor (VEGF) was present on cancer cells and stromal macrophages. These results demonstrate tumor cell-associated TF-dependent extravascular coagulation activation in situ in gastric cancer that does not appear to be counterbalanced by TFPI or sufficient fibrinolytic activity. Colocalization of VEGF with hemostatic proteins suggests that they may cooperate in the pathogenesis of gastric cancer.

Paracrine cyclooxygenase-2-mediated signalling by macrophages promotes tumorigenic progression of intestinal epithelial cells.

In human colorectal adenomas or polyps, cyclooxygenase-2 is expressed predominantly by stromal (or interstitial) macrophages. Therefore, we tested the hypothesis that macrophage cyclooxygenase-2 has paracrine pro-tumorigenic activity using in vitro models of macrophage-epithelial cell interactions. We report that macrophages can promote tumorigenic progression of intestinal epithelial cells (evidenced by decreased cell-cell contact inhibition, increased proliferation and apoptosis, gain of anchorage-independent growth capability, decreased membranous E-cadherin expression, up-regulation of cyclooxygenase-2 expression, down-regulation of transforming growth factor-beta type II receptor expression and resistance to the anti-proliferative activity of transforming growth factor-beta(1)) in a paracrine, cyclooxygenase-2-dependent manner. Pharmacologically relevant concentrations (1-2 microM) of a selective cyclooxygenase-2 inhibitor had no detectable, direct effect on intestinal epithelial cells but inhibited the macrophage-epithelial cell signal mediating tumorigenic progression. Cyclooxygenase-2-mediated stromal-epithelial cell signalling during the early stages of intestinal tumorigenesis provides a novel target for chemoprevention of colorectal cancer (and other gastro-intestinal epithelial malignancies, which arise on a background of chronic inflammation, such as gastric cancer) and may explain the discrepancy between the concentrations of cyclooxygenase inhibitors required to produce anti-neoplastic effects in vitro and in vivo.

Impaired splenic erythropoiesis in phlebotomized mice injected with CL2MDP-liposome: an experimental model for studying the role of stromal macrophages in erythropoiesis

Erythropoiesis occurs in the presence of erythropoietin (EPO) without macrophages in vitro. In hematopoietic tissues, however, erythroid cells associate closely with stromal macrophages, forming erythroblastic islands via interactions with adhesion molecules. To elucidate the role of macrophages in erythropoiesis, we selectively abrogated stromal macrophages of splenic red pulp of phlebotomized mice by injection with dichloromethylene diphosphonate encapsulated in multilamellar liposomes (CL2MDP-liposome). In the spleen, no erythropoietic activity occurred until 5 days after the treatment. Colony assay revealed that the erythropoiesis was suppressed at the level of CFU-E. The splenic erythropoietic activity gradually developed from day 6 after the treatment, when F4/80+ macrophages began to appear in the red pulp. EPO mRNA was expressed in kidney but not in liver or spleen of phlebotomized mice injected with CL2MDP-liposome, and the serum EPO concentration in these mice was higher than that in phlebotomized mice. These findings suggest that abrogation of stromal macrophages by injection with CL2MDP-liposome impairs the splenic microenvironment for erythropoiesis induced by hypoxic stress, and this may be an excellent experimental model for further characterization of the in vivo role of splenic macrophages in erythropoiesis.

Functional disturbance of marrow stromal microenvironment in the myelodysplastic syndromes.

The potential contribution of abnormal marrow stromal function to ineffective haemopoiesis in the myelodysplastic syndromes is unclear. We have compared the ability of stromal layers from normal (n = 7) and myelodysplastic (n = 9) marrow to alter proliferation and survival of the granulocyte-macrophage colony-stimulating factor/interleukin-3-dependent cell line F-36P. Co-cultures for 72 h in the absence of exogenous cytokines were either in direct contact with stroma or separated by transwell inserts. On normal stromal layers, the ratio of adherent F-36P cells relative to stromal cells increased from a mean of 0.2 +/- 0.01 (s.d.) at 4 h of co-culture to 0.34 +/- 0.08 after 72 h (n = 7). Corresponding values on myelodysplastic stroma (0.2 +/- 0.02 at 4 h and 0.35 +/- 0.05 at 72 h; n = 9) indicated that the ability of myelodysplastic stromal layers to regulate short-term proliferation of F-36P cells may be similar to normal. Apoptosis of F-36P cells was quantified after co-culture with normal or myelodysplastic stroma: results from myelodysplastic co-cultures were standardized as a fraction of values from co-cultures with paired normal stroma (apoptotic ratio). Augmented apoptosis of F-36P cells was detected in 8/9 co-cultures with myelodysplastic stroma (mean = 15.7 +/- 9.7%, n = 9), compared with corresponding normal stroma (mean = 12.4 +/- 4.6%, n = 7, P < 0.05) with a mean apoptotic ratio of 1.4 +/- 0.5 (P < 0.05). There was no correlation between stroma-related apoptosis and FAB type, tumour necrosis factor-alpha concentrations in the culture supernatant or numbers of stromal macrophages, and no evidence of involvement of the Fas pathway. Increased apoptosis was detected in cells grown in transwell inserts over stroma (23.8 +/- 3%, n = 5) compared to adherent cells in cultures with normal stromal layers, but this survival difference was not observed in co-cultures with myelodysplastic stroma. These results suggest that abnormal stromal function in patients with myelodysplastic syndromes may contribute to increased apoptosis of haemopoietic cells within the marrow microenvironment. The effect appears to be dependent on close cellular contact, rather than the release of soluble factors, but the exact mechanism remains unclear.

Expression of Oestrogen and progesterone receptors, Ki-67,p53 and bcl-2 proteins, cathepsin D, urokinase plasminogen activator and urokinase plasminogen activator-receptors in carcinomas of the female breast in an African population

To determine the expression of oestrogen (ER) and progesterone receptor (PgR), Ki-67, p53, bcl-2 proteins and the proteolytic enzymes cathepsin D (CD), urokinase-plasminogen activator (uPA) and its receptor (uPA-R) in primary carcinomas of the breast from indigenous Tanzanian female patients by immunohistochemistry. Prospective cross-sectional study. Muhimbili Medical Centre, Dar es Salaam, Tanzania. Sixty patients admitted between 1995 and 1997. Markers were found to be expressed as follows: ER (33.3%), PgR (18.3%), p53 (30%), bcl-2 (43.5%) and the median proliferation rate of Ki-67 was 15%. Proportion of tumours positive for ER, PgR and bcl-2 initially decreased to 12 months disease duration, after which it increased. The observed proliferation rate approaches that reported in developed countries. p53 expression did not influence the proliferation rate nor did bcl-2 expression. ER, PgR and bcl-2 were strongly co-expressed. CD was predominantly expressed in stromal macrophages than in cancer cells. The low expression of ER and PgR and their strong co-expression with bcl-2 might negatively influence response to hormonal therapy. The influence of bcl-2 on tumour response to anti-cancer therapy in patients with long disease duration requires urgent clarification. Determination of CD in stromal macrophages rather than in cancer cells may have greater prognostic significance in patients of this region.

Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors.

Serrate/Jagged and Delta are cell surface ligands for Notch receptors that may influence hematopoietic cell fate decisions and are known to be expressed in bone marrow stromal cells. In a series of screenings of cDNAs constructed by a cDNA library subtraction technique, we identified Jagged1, one of the Notch ligands, as a gene up-regulated by macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Therefore, we compared stromal cells and macrophages for expression of Notch ligands including Jagged1 and analyzed the regulation of their expression by cytokines. Murine bone marrow macrophages were prepared by culturing femoral bone marrow cells with M-CSF. Primary bone marrow fibroblastic stromal cells were prepared by a culture system that we recently developed. The expression of Notch ligands was analyzed by either Northern blot analysis or reverse transcriptase polymerase chain reaction. The bone marrow macrophages expressed Jagged1 but not Jagged2 and Delta1 at a level that was detectable by Northern blot analysis. Expression of the Jagged1 gene was markedly up-regulated by growth factors for the cells, i.e., M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Expression of Jagged2 and Delta1 seldom was affected by the stimuli. The primary bone marrow fibroblastic stromal cells, and murine stromal cell lines, such as PA6 and ST2, also expressed Jagged1 transcript, at levels comparable to the steady-state level in macrophages. However, expression of the Jagged1 gene was little affected when these cells were stimulated with fibroblastic growth factor and platelet-derived growth factor. We demonstrated that bone marrow macrophages as well as stromal cells constitutively produced Jagged1 and that the expression was markedly up-regulated by hematopoietic growth factors, M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. The results highlight the involvement of macrophages and these growth factors in hematopoietic cell fate decisions via the production of Jagged1.

Patients with non-immune chronic idiopathic neutropenia syndrome have increased splenic volume on ultrasonography.

Clinically detectable splenomegaly is rarely seen in patients with non-immune chronic idiopathic neutropenia syndrome (NI-CINS). Using ultrasound, we estimated splenic volume in 52 NI-CINS patients and 14 age- and sex-matched normal controls by determining the "corrected splenic index" (CSI) from the product of length, width and thickness of the organ expressed in cm3/m2 body surface area. We found that CSI was significantly higher in the group of patients compared to controls (202.8 +/- 82.0 vs. 133.8 +/- 28.1 cm3/m2, P=0.003), and that individual CSI values was inversely correlated with the number of circulating neutrophils (r=-0.5097, P < 0.0001). About 48.1% of the patients had CSI above 190 cm3/m2 body surface, representing the upper 95% confidence limit of values found in the controls. Patients also had increased serum concentrations of pro-inflammatory cytokines and chemokines mainly produced by activated macrophages (IL-1beta, TNF-alpha, RANTES and IL-8), as well as increased serum levels of soluble cell adhesion molecules derived from activated endothelium (sE-Selectin, sICAM and sVCAM). We hypothesize that the increased splenic volume in NI-CINS patients may be due to the accumulation of activated macrophages inside the spleen, possibly as the result of an unrecognized low-grade chronic inflammatory process. The nature of such an inflammation is unknown. A study was designed to search for viral or bacterial genomic material in patients' bone marrow stromal macrophages in which the unknown causal agent might be located.

NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps.

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-kappaB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-kappaB is seen in stromal macrophages that also express COX-2 and TNF-alpha, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-kappaB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

Expression of monocyte chemoattractant protein-1 in peritoneal endometriotic cells.

It is well known that the number of peritoneal macrophages is increased in patients with pelvic endometriosis. We measured the concentration of monocyte chemoattractant protein-1 (MCP-1) using an enzyme-linked immunosorbent assay (ELISA) in the peritoneal fluid of women with and without endometriosis. The expression of MCP-1 in pelvic endometriotic lesions obtained from the peritoneum was also examined using immunohistochemistry and nonradioactive in situ hybridization. The mean concentration of MCP-1 in the peritoneal fluid was significantly higher in the patients with endometriosis (P<0.05). The most significant elevation, compared with non-endometriosis patients, was found in stage I of the disease (P<0.05). However, no statistically significant difference was found among endometriosis stages I, II, III, and IV. Immunohistochemical staining revealed that MCP-1-positive cells were localized in the glandular epithelium of the endometriotic lesions and in the stromal macrophages distributed in those lesions, but normal peritoneal cells were negative. The in situ hybridization method demonstrated expression of MCP-1 mRNA on the endometriotic glandular epithelium and stromal macrophages. These findings suggest that MCP-1 may be involved in the histogenesis and early development of peritoneal endometriosis.

In vivo production of cytokines by bone marrow stromal cells and macrophages from patients with myelodysplastic syndrome.

We studied functional disturbances in hemopoietic microenvironment and cytokine production by stromal sublayer in long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome. Production of factors stimulating the growth of normal erythroid and granulocytic precursors by cells of the stromal sublayer from patients with refractory sideroblast anemia and refractory anemia with excess blasts is impaired compared to cells from healthy donors. The medium conditioned by macrophages from patients with chronic myelomonocytic leukemia displayed a higher ability to stimulate the growth of granulocytes and macrophages compared to media conditioned by cells from donors and patients with refractory sideroblast anemia and refractory anemia with excess blasts. Cultured stromal cells and macrophages produced tumor necrosis factor-alpha and interleukin-6. Their content in media conditioned by cells from patients with myelodysplastic syndrome surpassed that in healthy donors. Our results suggest that production of cytokines by stromal microenvironmental cells is impaired in patients with various forms of myelodysplastic syndrome.

Effects of Vascular Endothelial and Platelet-derived Growth Factor Receptor Inhibitors on Long-term Cultures from Normal Human Bone Marrow

Endothelial cells and fibroblasts are important constituents of the haemopoietic microenvironment. Growth and function of these cells are controlled by a variety of cytokines, including vascular endothelial growth factor (VEGF) and plateletderived growth factor (PDGF). We analysed the effects of novel tyrosine kinase inhibitors targeting the VEGF and PDGF receptors (compounds SU5614 and SU5768) on the performance of long-term cultures from normal human bone marrow.In developing cultures, the inhibitors induced a dosedependent reduction in stromal fibroblasts, macrophages and endothelial cells with a concomitant decrease in blood cell production and an increase in fat cells. For SU5614, the concentration inhibiting stroma formation by 50% (IC50) was 123 nM, and the IC50 for haemopoietic colony forming cell output was 186 nM. For S SU 5768, the respective values were 871 nM and 331 nM. Changes in stroma composition and inhibition of haemopoietic cell production were also demonstrable after delayed addition of the inhibitors to established cultures. By contrast, haemo-poietic colony formation in clonogenic agar cultures was unimpaired (IC50 not reached at 100 μM). Immunofluorescence studies and time course analyses suggested that the primary effect of the inhibitors was interference with the proliferation and function of fibroblasts and endothelial cells which in turn resulted in decreased haemopoiesis and increased adipogenesis. This was associated with decreased levels in conditioned media of granulocyte-macrophage colony-stimulating factor, interleukin-6 and leptin.VEGF and PDGF may play a hitherto underestimated role in the control of blood cell formation. VEGF/PDGF receptor inhibitors may have therapeutic potential in stroma diseases such as myelofibrosis. Since they weaken the stimulatory signals provided by the microenvironment, they may also be of value in the treatment of leukaemia and other neoplastic bone marrow diseases.