Direct monoclonal antibody rosetting. An effective method for weak antigen detection and large scale separation of human mononuclear cells.

A direct monoclonal antibody rosetting technique is described which serves as a simple, reliable, and very sensitive method for the detection of surface antigens on human leukocytes. In this technique the discriminative monoclonal anti-leukocyte antibodies are directly coupled to ox erythrocytes (oxE) by use of CrCl3. The procedure can be applied to the effective separation of mononuclear cell subsets. By choosing a gradient of appropriate density labeled cells are either isolated in high purity or quantitatively eliminated. This technique proved to be particularly suitable for the large scale purification of autologous or allogeneic bone marrow transplants where either leukemic cells or T cells have to be completely eliminated without damaging sensitive stem cells.

Recovery of monocytes after bone marrow transplantation--rapid reappearance of tumor necrosis factor alpha and interleukin 6 production.

After bone marrow transplantation many T-lymphocyte functions, including the production of cytokines (CK), such as interleukin 2, are severely depressed for months. The monocyte-derived cytokines tumor necrosis factor alpha and interleukin 6 are molecules central to immune functions. Moreover, they may be involved in graft-versus-host disease and in graft-versus-leukemia reaction. Hence, we have studied the reappearance of these CKs after BMT by analyzing whole blood cultures stimulated in vitro with lipopolysaccharide for 6 hr, followed by testing for the secretion of TNF in the WEHI 164/actinomycin D cytotoxicity bioassay and for IL-6 in the 7 TD 1 proliferation assay. We performed sequential studies in 6 children who were transplanted for aplastic anemia or leukemia with allogeneic bone marrow. We found that the production of both CKs can be induced as early as 10-14 days post BMT at the very beginning of engraftment, indicating that the regenerating monocyte system is recovering rapidly after BMT. Depletion and neutralization experiments confirmed that monocytes are the cellular source of the LPS-induced CK secretion after BMT. Control levels were reached 3 to 4 weeks post BMT. When analyzing the endotoxin-induced CK production in a larger panel of BMT patients after complete reconstitution, we could not detect any impact of acute or chronic GvHD, or of allogeneic or autologous BMT, nor did treatment with cyclosporine A (CsA) show any suppressive effect. Thus, our data show that the CK production of the monocyte/macrophage lineage is quite resistant to factors that do influence other cell lineages of the immune system during BMT. The coincident appearance of monocyte-derived cytokines and of GvHD suggests a role for these cytokines in GvHD in man.

Interleukin-10 is upregulated in LPS tolerance.

Lipopolysaccharide (LPS) stimulation of the human monocytic cell line Mono Mac 6 leads to rapid expression of both the pro-inflammatory cytokine tumor necrosis factor (TNF) and the anti-inflammatory cytokine interleukin-10 (IL-10). Preculture of these cells with a low dose of LPS for 2 days rendered the cells tolerant to subsequent stimulation, in that TNF gene expression is only minimal, both at the mRNA and at the protein level. IL-10 shows a reciprocal pattern, however, as expression of this gene is upregulated in precultured cells, and it will further increase upon subsequent stimulation. Although TNF has been shown to induce IL-10, and IL-10 was found to downregulate TNF, this reciprocal regulation does not explain the pattern observed in LPS tolerance in Mono Mac 6, since neutralizing antibodies against TNF and IL-10 could not prevent upregulation of IL-10 and downregulation of TNF, respectively. Treatment of Mono Mac 6 cells during LPS preculture with interferon-gamma (IFN-gamma) could, however, reverse tolerance: LPS/IFN-gamma precultured cells produced high levels of TNF transcripts upon subsequent stimulation, while the response of the IL-10 gene was attenuated. The data show that LPS tolerance does not involve a passive downregulation of all types of monocyte functions, but it is an orchestrated response with downregulation of pro- and upregulation of anti-inflammatory cytokines.

Interleukin-6 (IL-6) levels in febrile children during maximal aplasia after bone marrow transplantation (BMT) are similar to those in children with normal hematopoiesis.

Interleukin-6 (IL-6) has been shown to be an inducer of the acute-phase response (APR) and to be involved in the pathogenesis of several disease states, including graft-versus-host disease (GvHD) following allogeneic bone marrow transplantation (BMT). As blood cells of the monocyte lineage are known to be major producers of this cytokine, we wondered whether extreme peripheral leukopenia following total ablation of hematopoiesis could compromise IL-6 production during the first days after allogeneic or autologous BMT. In the absence of detectable circulating leukocytes we measured elevated IL-6 levels in six children having fever (> or = 38 degrees C) of presumed infectious origin with an average of 74 +/- 60 units/ml (range 19-309 units/ml). IL-6 levels in febrile children having a normal hematopoiesis (118 +/- 254 units/ml, range 17-1213 units/ml) were not significantly higher than those found in the febrile BMT group (p > 0.05). Moreover, there was a clear association between elevated IL-6 levels and the presence of fever. C-reactive protein (CRP) was also elevated (> or = 1 mg/dl), whereas tumor-necrosis factor alpha (TNF) was undetectable (< 1 pg/ml). Two transplanted patients without fever during the period of total aplasia had neither detectable CRP nor IL-6, thus demonstrating that the transplant procedure itself does not induce an APR. Our data obtained during maximal leukopenia following BMT show that a functional hematopoietic system is not necessary for regular production of IL-6, which is associated with fever. Cells of nonhematopoietic origin may contribute to this production.

Detection of neuron-specific gamma-enolase messenger ribonucleic acid in normal human leukocytes by polymerase chain reaction amplification with nested primers.

BACKGROUND: NNE (non-neuronal alpha-enolase) is a glycolytic enzyme detected in most tissues. NSE (neuron-specific gamma-enolase) is detected in normal neurons and tumors such as neuroblastoma. Staining with antibodies against NSE is therefore used to detect neuroblastoma cells invading bone marrow. Since staining of normal leukocytes has been reported we asked whether bona fide NSE is in fact expressed in normal blood and marrow. EXPERIMENTAL DESIGN: We designed nested coding region specific primers for NSE and NNE and, after reverse transcription of mRNA, we amplified the coding region between these primers in a semi-nested polymerase chain reaction. In order to distinguish both iso-mRNAs from each other, we amplified a long (1,047 bp) template in a first round of 30 cycles with primers specific for NNE or NSE. One percent of this product was used in a second round of 30 cycles in which both sense primers and two nested anti-sense primers of alternate specificities yielding shorter products of discernible sizes (768 bp or 619 bp) were added together in the same reaction tube. With this combination of four primers, only that shorter product was amplified to visibility, the specificity of which was homologous to the template produced in the first 30 cycles. Restriction enzyme digestion of the amplified products was used to verify this polymerase chain reaction-based approach for the distinction of isoforms of RNA. RESULTS: This semi-nested polymerase chain reaction clearly allows for the distinction of mRNA for NNE or NSE and shows the presence of transcripts for NSE in normal human leukocytes from blood and bone marrow. CONCLUSIONS: This method exploiting short stretches of nucleotide differences in the coding regions for priming can more generally be applied to the distinction of all isoforms of RNA where nested specific primers can be designed. However, the presence of NSE specific transcripts in normal human leukocytes invalidates the use of this highly sensitive method as a disease marker in neuroblastoma.

Differential cytokine expression in human blood monocyte subpopulations: a polymerase chain reaction analysis.

The subpopulation of strongly CD14-positive (CD14++) monocytes and monocytes coexpressing the CD16 antigen and low levels of CD14 (CD14+/CD16+ cells) were isolated by fluorescence-activated cell sorting (FACS) followed by stimulation with lipopolysaccharide (LPS) at 1 micrograms/mL. Polymerase chain reaction (PCR) after reverse transcription of isolated mRNA (RT-PCR) revealed similar levels of tumor necrosis factor (TNF) transcripts in both subpopulations. By contrast, transcripts for interleukin-10 (IL-10) were only detectable in CD14++ monocytes, whereas CD14+/CD16+ cells produced no detectable IL-10 transcripts after 4 hours. Only after 16 hours of LPS stimulation was a low level of IL-10 transcripts discernible in CD14+/CD16+ monocytes. The same pattern was seen at the protein level in that TNF in LPS-stimulated supernatants was comparable for both subpopulations, whereas IL-10 was detected in CD14++ monocytes but not in CD14+/CD16+ cells. To avoid interference of cell activation by CD14 and CD16 antibodies, cells were also isolated based on the high and low level of CD33 antigen expression. Again, weakly CD33-positive cells, which comprise the CD14+/CD16+ cells, showed no or only minimal IL-10 mRNA. When comparing blood monocyte subpopulations with alveolar macrophages (AM), AM showed high levels of LPS-stimulated TNF, whereas IL-10 transcripts were undetectable. Our data show that CD14+/CD16+ blood monocytes produce high levels of proinflammatory cytokines like TNF, whereas the anti-inflammatory IL-10 is low or absent, a pattern similar to what is seen in AM.

CD14 is expressed and functional in human B cells.

The human B cell line RPMI 8226 exhibits variable staining with the CD14 antibody My4. We have isolated three stable clones from this line with clones 1 and 2 being My4 positive and clone 3 My4 negative. Similar to previous results in monocytes, immunoprecipitation with the My4 antibody revealed a 54-kDa cell surface molecule, analysis of supernatants showed soluble CD14, and Northern blotting demonstrated a 1.4-kb transcript in clones 1 and 2, but not in clone 3, which suggests that the My4 antibody detects CD14 in clones 1 and 2. This CD14 molecule was functional in that lipopolysaccharide stimulation induced interleukin (IL)-6 and IL-10 in clones 1 and 2 but not in clone 3. Furthermore, the My4 antibody was capable of blocking these responses at the transcript and protein levels. Finally, peripheral blood B cells were highly purified by cell sorting (> 98% CD19 positive). These cells produced IL-6 in response to lipopolysaccharide, and this response was blocked by anti-CD14 antiserum. Thus, our findings demonstrated that human B cells can express functionally active CD14.

[Juvenile chronic leukemia and chronic myelomonocytic leukemia. Experiences with bone marrow transplantation in childhood in 5 cases]. / Juvenile chronische Leukamie und chronische myelomonozytäre Leukämie. Erfahrungen mit der Knochenmarktransplantation im Kindesalter anhand von 5 Fällen.

Chronic leukemias of early childhood (JCML/CMML) are rare malignant diseases for which effective regimens of chemotherapy have not been established. However, some of these patients may be cured by BMT. We report on 4 children with JCML and one child with CMML undergoing BMT from HLA-identical siblings and from matched unrelated donors. 3 of 5 patients are disease-free 9 to 37 months post BMT. According to our observations an effective reduction of blastic cells before BMT seems to be necessary for a sustained remission of these diseases. This reduction can be reached by intensive chemotherapy and by splenectomy before BMT. Moreover, we stress the need for total body irradiation (TBI) during BMT in order to eradicate residual malignant cells.

Increased serum levels of tumor necrosis factor alpha precede major complications of bone marrow transplantation.

Acute graft-versus-host disease, interstitial pneumonitis, endothelial leakage syndrome, and veno-occlusive disease are major complications of bone marrow transplantation. Though several new regimens for prophylaxis and treatment of these syndromes have been introduced, the overall incidence has been only slightly reduced over the last few years. We retrospectively analyzed tumor necrosis factor alpha (TNF alpha) serum levels between day -8 and day 100 after bone marrow transplantation in 56 patients transplanted in our unit for a variety of hematological diseases. In 34 patients with uneventful courses, mean TNF alpha levels rose to a maximum of 76 +/- 29 pg/mL. In contrast, 22 patients with major transplant related complications showed mean increases of TNF alpha of 492 +/- 235 pg/mL (P less than .0001). Increases of TNF alpha occurred before interstitial pneumonitis and severe acute graft-versus-host disease with a latency of 25 to 54 days. Early complications such as endothelial leakage syndrome and veno-occlusive disease were closely associated with increases of TNF alpha serum levels. Our study suggests two pathways of TNF alpha release: activation of host macrophages and stimulation of donor cells in the course of acute graft-versus-host disease. Cytokine monitoring should be helpful for prediction and earlier treatment of major transplant related complications.