Introduction
Polymorphonuclear neutrophils (PMNs) constitute about 75% of the white blood cells of normal individuals’ peripheral blood and are the major component (>90%) of the granulocytes. They take part in the acute inflammatory response and are the primary immune effector cells against extracellular pathogens. They can be activated by macrophage- and endothelial- derived cytokines. Neutrophils express Fc receptors for IgG and complement receptors and thus bind and phagocytose opsonized antigens, providing an important link between innate and humoral immunity (Pure, 2000). PMN has got special importance in hematological malignancies as the majority of patients of all types show infectious complications associated with neutropenia in AML (Estey et al, 2000), ALL (Dubansky et al, 1989; Benz et al, 2000; Hoelzer, 2000), CML (Crammer et al, 1977), and MDS (Greenberg, 2000). It has been reported that functional activities of PMNs were defective in ALL children undergoing chemotherapy (Lejeune et al, 1998). In addition, some morphological alteration in PMN was also observed in CML and MDS cases. In this connection, it may be noted that both CML and MDS are being regarded as a clonal hematopoietic progenitor cell disorder, and therefore it may be interesting to study the status of PMN in these disorders. Furthermore, it has been established that PMN could be activated for the treatment of B-lymphoblastoid malignancies (Valerius et al, 1997), and is more effective against IL-2 transfected tumor cells (Pericle, 1996) and Rituximab directed CD20+ B-cells (Hernandez-Ilizaliturri, 2003). Evidence also suggests that PMN has direct anti-tumor cytotoxic effect (Koga, 2004). Therefore, it would be interesting to find the HLA status (both class I and II) to get an idea about the pathophysiological state of the first line of defense mechanism of the immune system under the condition.
Materials and Methods
Patients and Normal Individuals
10 ml Peripheral blood (PBL) or 2 ml of bone marrow (BM) samples were collected as per institutional ethical guidelines at the time of diagnosis (de novo & untreated) from the patients suffering from different hematological malignancies. The diagnosis and immunophenotyping categorization of different leukemic patients were done in accordance with the French-American-British (FAB) as mentioned earlier (Majumder et al, 2005; Majumder et al, 2006). Thus, samples of 15 acute myeloid leukemia (AML) (2 of them are M4 and the rest of the samples are either M1 or M2, immunophenotyping characterization also confirms the light microscopic observation), 14 acute lymphoid leukemia (ALL) (all are L1 or L2; immunophenotyping characterization was either CD19+ or CD19+ and CD20+ B lineage cell), 16 chronic myeloid leukemia (CML) (all are in chronic phase and are Ph+), 8 chronic lymphoid leukemia (CLL) (all are either CD19+ or both CD19+ and CD20+ CLL) and 8 myelodysplastic syndrome (MDS) (3 are refractory anemia-MDS, 4 are AML-MDS) were used in this study. Peripheral blood was also collected from 15 normal healthy volunteers (NV) following institutional ethical guidelines.
Processing of Samples
Briefly, collected samples were first diluted about four times and then separated into two compartments by layered preformed Percoll (Sigma, USA) density gradient centrifugation at a density of 1.077 and 1.093 (Pertoft and Lakarent, 1982). Mononuclear cells at 1.077 were removed first, then cells at 1.093 were processed further by using CD15-MACS microbeads and MiniMACS column (Miltenyi Biotec GmbH, Germany) for the enrichment of CD15+ cells (positive panning procedure). Cells isolated at 1.077 were processed further to separate leukemic/immature cells as described earlier (Majumder et al, 2005).
Cell Lines
Raji cell line was obtained from the National Centre for Cell sciences (Pune, India) and was maintained in 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (GIBCO-BRL, USA). This has been used as a positive control for flow cytometric analysis of HLA-ABC and HLA-DR (Majumder et al, 2005).
Analysis of Purification of CD15+ Cells by Flow Cytometry
For the evaluation of the purification of neutrophils, enriched cells (as mentioned in processing of samples) were stained with fluorescent isothiocyanate (FITC) conjugated anti-CD15 (BD 340703), phycoerythrin (PE) conjugated anti-CD8 (BD 340046) and allophycocyanin (APC) conjugated anti-CD34 (BD 340667) and subjected to flow cytometric analysis. Cells are gated (G1) first from FSC vs SSC and then analyzed for CD15 vs SSC, CD15 vs CD34 and CD15 vs CD8 by comparing against the corresponding isotype.
Flow Cytometric Analysis of CD15+ Cell Surface HLA-ABC and HLA-DR
For HLA-ABC and HLA-DR analysis in CD15+ cells’ surface, only fresh samples having more than 20% phenotypic neutrophils in their differential count were used. 100µl of the whole blood was stained with anti-CD15-FITC and anti-HLA-ABC-APC (BD 555555) and analyzed for HLA-ABC expression. Anti-CD15 was used for flow cytometry based phenotypic selection of neutrophils. Similarly, HLA-DR expression analysis on the CD15+ cell 100 ïl of the whole blood was incubated separately with anti-CD15-PE (SC-19595PE) together with anti-HLA DR-FITC (BD 347363) and analyzed by flow cytometer (FACSCalibur, Becton Dickinson, USA). For data analysis, neutrophilic gate (G1) was selected first from FSC and SSC, then from G1 another gate of CD15 vs SSC (G2) was constructed against the corresponding isotype so that only bright cells can be selected (except MDS cases) and the G2 cells were further analyzed for HLA-ABC and HLA-DR expression. Though there is little chance, however, with this method if leukemic blast cells having aberrant CD15 expression are selected then they may slightly skew the observation.
In each case, data of at least 3000-10000 CD15+ cells (depending on the case) were analyzed. FITC, PE, and APC conjugated antibody data were analyzed at Fl1, Fl2 and Fl4 respectively in the flow cytometric analysis. All monoclonals, isotypes, fixation buffer, and FACS Lysing solution were purchased from Becton Dickinson (BD), USA. All antibodies were used as per the manufacturer’s instruction.
Analysis of HLA-DR Activation on the CD15+ Granulocytes of NV in Presence of Primary Leukemic Cells
CD15+ granulocytes of NV were mixed with isolated leukemic cells [having immunophenotyping either CD33+ and CD15- (for AML) or CD19+ and CD15- (for ALL)] from leukemic patients in 2 ml of RPMI-1640 and were incubated at 37 ï‚°C with the effector to target cells ratio of 10:1 (Effector = 1ï‚´106 and target = 1ï‚´105 cells) for 20 minutes time interval (Vella et al, 2002). After incubation, cells are labeled with anti-HLA-DR-FITC and anti-CD15-PE (SC-19595PE) and analyzed by flow cytometry. FITC and PE conjugated antibody, data are analyzed at Fl1 and Fl2 respectively in the FACS analysis. CD15+ cells are gated (G1) first from the CD15 vs FSC compared to the isotype and analyzed for HLA-DR expression both in the control (without any target) and in the test (effector with the target) (Figure 3A).
Assessment of CD15+ Granulocytes’ Oxidative Burst Activity Against the Primary Leukemic Cells
To assess the oxidative burst activity (or by ROS production) of the CD15+ granulocytes towards the primary leukemic cells, 5ï‚´106 CD15+ cells of NV were co-cultured with 5ï‚´105 target cells [HLA downregulated leukemic cells: CML (HLA-A+ and -C+, but HLA-B-) and/or CD33+ or CD19+ (both has MFI of HLA-ABC<30)] as well as K-562 and Raji cells] in different tubes (test). The procedure of malignant cells isolation and the assigning of HLA downregulation was described earlier (Majumder et al, 2005). For each set of co-culture reaction, a control tube containing equal number of effector cells and a blank control tube containing equal number of target cells were also taken. To each tube, 200 ïl of 1% NBT (nitroblue tetrazolium, SRL, India) solution was added and volume made up to 3ml with RPMI-1640 and incubated at 37 ï‚°C for 6 hours. After incubation, reaction was stopped by adding chilled 0.1N HCl, centrifuged at 1000g for 15 minutes, supernatant was discarded and the pelleted blue formazone was extracted with equal volume of pyridine (SRL, India) and absorbance of the solution was taken spectrophotometrically at 488 nm (Hudson and Hay, 1993; De Toni et al, 1997; Law et al, 2001). In each of the respective cases, blank control was used to set zero in the spectrophotometer, and killing efficacy was evaluated as the differences in absorbance of the test and the control.
Statistical Analysis
Differences in HLA expression were analyzed by (two-sided) Mann-Whitney U test. A P value less than or equal to 0.05 was considered significant.
The role of immunological surveillance against tumors was largely focused with the lymphocytes and macrophages. Moreover, it has been suggested previously that T cell based immunotherapy will not provide any solution to the HLA downregulated cases. Surprisingly, the functionality of neutrophils against primary leukemic cells has not been documented. Recently, several mechanisms about its functionality have been suggested which include antibody dependent cell cytotoxicity (ADCC) (Elasser et al, 1996; Wurflein et al, 1998), Fas ligand mediated apoptosis (Kim et al, 2000), TNF (tumor necrosis factor) related apoptosis inducing ligand (TRAIL) mediated cytotoxicity (Koga et al, 2004), direct cell killing by phagocytosis followed by intracellular H2O2 and superoxide generation (Lichtenstein and Kahle, 1985). We found that CD15+ cells isolated from NV have a direct killing capacity of HLA downregulated primary leukemic cells, and also have the ability to express HLA class II in presence of primary leukemic cells. In this study, the enhanced HLA-DR expression by CD15+ cells of leukemic patients may be due to the constant cell contact with the malignant cells within the hematopoietic system. This work and our previous work confirm these observations (Majumder, 2012).
It is well established that leukemic patients of all types are more prone to the secondary infection (Estey et al, 2000; Dubansky et al, 1989; Benz et al, 2000; Crammer et al, 1977; Greenberg, 2000). Therefore, CD15+ cells transplantation to leukemic patients (either allogenic or autologous, depending on the case) may have a beneficial role, not only in preventing the secondary infection, but also in killing HLA downregulated primary leukemic cells by ROS production (Hudson and Hay, 1993). It may also be noted that recent clinical trials involving cord blood transplantation in hematological malignancies indicated a better outcome with cells containing a higher neutrophilic counts (Ishikawa and Miyazaki, 2005). Moreover, due to its capacity to express HLA-DR, a long-term benefit of T cell priming by PMNs would be able to provide an added advantage. Thus, as suggested previously (Arcese et al, 2006), the therapeutic application of CD15+ cells may bridge the innate and adaptive immunity in the state of hematological malignancies. Finally, our previous work and that of others (Polakova et al, 2003; Majumder et al, 2006) suggest that innate immune mechanism may be harped in to produce a positive supportive treatment for leukemia.
Acknowledgement
We are grateful to the clinicians and pathologists working in the Ramakrishna Mission Seva Prathisthan, Kothari Medical Center, Kolkata for collecting samples and clinical data. DM acknowledges University Grants Commission, INDIA for providing SRF (NET) Fellowship.
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