Release of CXC-chemokines by human lung microvascular endothelial cells (LMVEC) compared with macrovascular umbilical vein endothelial cells.

In the present study, the sensitivity of LMVEC and human umbilical vein endothelial cells (HUVEC) to lipopolysaccharide (LPS) and the proinflammatory cytokines IL-1, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) was compared. To this end, the production of the CC- (MCP-1), CXC- (IL-8, ENA-78, Groalpha, NAP-2, GCP-2) and CX3C (fractalkine) chemokines was studied. A low basal production of these chemokines was observed in both cell types. TNF-alpha, IL-1 and LPS up-regulated all chemokines tested. IFN-gamma however was only able to up-regulate MCP-1 production. LMVEC were more sensitive to IL-1 and LPS compared with HUVEC, since LMVEC produced significantly more MCP-1, ENA-78 and Groalpha (P < 0. 01) under these conditions. Maximal production of MCP-1 in LMVEC was achieved with TNF-alpha (28.4 ng/ml, P < 0.01), whereas IL-1 was the most potent stimulator of ENA-78 (2.78 ng/ml, P < 0.001) and Groalpha (29.2 ng/ml, P < 0.001). IL-8 production in LMVEC cells was maximal after LPS stimulation (28.4 ng/ml), but lower than on HUVEC (P < 0.01). LPS, TNF-alpha and IL-1 stimulation strongly up-regulated all chemokine mRNA. No quantitative differences in mRNA expression between LMVEC and HUVEC were detected for MCP-1 and Groalpha after LPS stimulation. mRNA expression of ENA-78, GCP-2, CX3C and NAP-2 was however higher in LMVEC under LPS stimulation. In contrast, IL-8 mRNA was slightly more expressed in HUVEC under these conditions. These results further support the hypothesis that the microvascular lung endothelium plays an active role in the induction and perpetuation of acute lung injury.

Iododeoxyuridine labelling of S-phase fraction in fine needle aspirates from breast carcinomas.

The suitability of measuring the S-phase fraction in human breast cancer by labelling tumour cells from fine needle aspirates (FNAs) in vitro with iododeoxyuridine (IdU) was studied in 11 patients. The S-phase fraction was measured both in preoperative FNAs labelled in vitro with IdU, and in FNAs taken from the same tumour when surgically removed after intravenous administration of IdU. Frozen sections were also immunostained for incorporated IdU. The mean S-phase fraction measured in FNAs after in vitro or in vivo labelling and in sections after in vivo labelling was 4.0, 3.6, and 3.1, respectively. Results of in vitro and in vivo labelling of FNAs with IdU were similar. However, as the S-phase fraction in breast cancer is generally low, the variation between the different measurements is too large; therefore, the S-phase fraction is not a suitable indicator of response to treatment.

Growth arrest associated changes of mRNA levels in breast cancer cells measured by semi-quantitative RT-PCR: potential early indicators of treatment response.

To find early and sensitive indicators of treatment response in breast cancer, we studied the mRNA levels of proliferation-related genes during growth arrest of the human breast cancer cell lines T47D and MCF7. A sensitive reverse transcriptase-PCR (RT-PCR) technique was used in order to monitor gene expression in small samples of cells. Estrogen-depletion and treatment with tamoxifen effectively induced a G1-arrest in both cell lines, accompanied by a decrease of the mRNA levels of histone H4, cyclin A, cyclin D1, and c-myc. Cyclin A expression decreased most strongly: up to 32-fold within 7 days. The expression of c-fos and WAF1 increased during growth arrest. In conclusion, significant changes of the levels of proliferation-related mRNAs, induced by growth arrest, can be measured in small samples of breast carcinoma cells using RT-PCR. Especially the decrease of the cyclin A mRNA level seems a potential early indicator of clinical response to tamoxifen therapy in breast cancer patients.

Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates.

BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA). EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR. RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells. CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.

Migration of dendritic cells into the draining lymph nodes of the lung after intratracheal instillation.

The migration of dendritic cell (DC)-enriched populations and alveolar macrophage (AM) populations isolated from PVG RT7.2 rats was studied after local administration to recipient PVG RT7.1 rats. The monoclonal antibody His41, which is directed against the common leukocyte antigen of the RT7.2 rat, was used to detect migrated cells. Injection of the splenic DC and AM subcutaneously into the footpads resulted in migration of both cell types to the popliteal lymph nodes after 24 h. DC located predominantly in the T cell-dependent areas, whereas AM located more in the medulla and medullary cords and spread throughout the outer cortex area. After intratracheal instillation of splenic DC, these cells were found predominantly in T cell-dependent areas of the draining lymph nodes of the lung after 24 h. In contrast, AM did not migrate to the draining lymph nodes after intratracheal instillation. Combined with those from earlier studies, these data show that DC present in the alveolar lumen may pick up airborne antigen and migrate to the draining lymph nodes of the lung, where they can induce primary T cell responses.

Separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional characterization.

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.

T cell priming in situ by intratracheally instilled antigen-pulsed dendritic cells.

In the present study, splenic dendritic cells (DC) and alveolar macrophages (AM) were pulsed with antigen in vitro and subsequently intratracheally instilled to test whether these cells have the capacity to sensitize T cells in the draining lymph nodes of the lung. The data demonstrate that antigen-pulsed DC, instilled in the bronchoalveolar lumen, induce antigen-specific T cell priming in vivo in the draining lymph nodes. T cell priming is only seen with viable but not with killed antigen-pulsed DC. Amounts as low as 5 x 10(3) to 10 x 10(3) cells can still induce some responsiveness. In addition, it was found that instillation of viable as well as killed pulsed Ia-negative AM also leads to T cell priming, although about 10 times higher numbers of cells had to be used in comparison with DC. The results suggest that DC instilled in the bronchoalveolar lumen present antigen directly to naive T cells, whereas for AM other mechanisms are involved.

An improved and rapid method for the isolation of rat lymph node or spleen T lymphocytes for T cell proliferation assays.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.

Effect of Bacillus Calmette-Guérin inoculation on numbers of dendritic cells in bronchoalveolar lavages of rats.

Dendritic cells (DC) are present in all lymphoid tissues and are widely distributed in the airway epithelium and lung parenchyma. In this study DC were morphologically and cytochemically identified in normal rat bronchoalveolar lavage (BAL), although in very low percentages. Furthermore, the total population as well as different Percoll density fractions demonstrated poor antigen-presenting capacity and even suppressed antigen-specific stimulation by rat splenic DC. In contrast, when an inflammatory response was induced by intratracheal inoculation of Bacillus Calmette-Guérin (BCG), an increase of Ia-positive cells, containing high percentages of monocytes and DC (MDC) was found. In BAL, DC increased about 25 times within 48 h after BCG inoculation. These BCG-induced BAL cells as well as the different density fractions showed a high antigen-presenting capacity at low concentrations. However, at higher concentrations they were suppressive, except for the highest density fraction which lacked alveolar macrophages (AM). These results indicate that the increased numbers of Ia-positive MDC during an inflammatory reaction are very likely responsible for antigen presentation in vitro. In contrast, AM suppress the antigen-specific T cell proliferation in a concentration dependent manner.