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[Cancer Research 60, 5420-5426, October 1, 2000]
© 2000 American Association for Cancer Research

Clinical Investigations

Reconstitution of Endogenous Interferon {alpha} by Recombinant Interferon in Hairy Cell Leukemia1

Medhat Shehata, Josef D. Schwarzmeier2, Son Tho Nguyen, Martin Hilgarth, Rudolf Berger, Rainer Hubmann, Sonja Kickmaier and Thomas Decker

University of Vienna, Clinic of Internal Medicine I, Department of Hematology, [M. S., J. D. S., M. H.], and L. Boltzmann Institute for Cytokine Research [M. S., J. D. S., S. T. N., R. B., R. H., S. K.], University of Vienna, A-1090 Vienna, Austria, and Institute for Microbiology and Genetics, A-1030 Vienna, Austria [T. D.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant human IFN {alpha} (rhIFN-{alpha}) plays an important role in the treatment of hairy cell leukemia (HCL). However, the mechanisms leading to its beneficial effect are not completely clarified, and there is no information on IFN-{alpha} gene expression in this disease. Therefore, we investigated the pattern of IFN-{alpha} gene expression and protein production in HCL and their potential regulation by rhIFN-{alpha}. Blood samples from 10 patients with HCL and 8 healthy donors (HD) were investigated. Expression of IFN-{alpha} mRNA was assessed by reverse transcription-PCR analysis in peripheral blood mononuclear cells (PBMCs) under basal conditions and on induction with rhIFN-{alpha} and polyionosinic-polycytidylic acid [poly(I·C)]. IFN-{alpha} concentrations in plasma and culture supernatants were measured by immunoassays, and intracellular IFN-{alpha} was evaluated by fluorescence-activated cell sorting analysis. Results showed that, in contrast to blood samples from HDs, freshly isolated PBMCs from untreated HCL patients did not express IFN-{alpha} mRNA, whereas IFN-{alpha} transcripts were found in patients who were under rhIFN-{alpha} therapy. Plasma of untreated patients contained no, or extremely low levels of, IFN-{alpha} as compared with plasma of treated patients and HDs. Ex vivo treatment of PBMCs with rhIFN-{alpha} or poly(I·C) resulted in a remarkable up-regulation of IFN-{alpha} at the mRNA and protein level. In HCL, however, the amounts of IFN-{alpha} protein remained less than in HD. Inhibition of IFN-{alpha} transcription was found after exposure of PBMCs to serum from untreated patients. Finally, a reduced capacity to produce IFN-{alpha} was found within B- cell, T-cell, and monocyte compartments in HCL patients, which could be enhanced by rhIFN-{alpha}. The results demonstrate the ability of rhIFN-{alpha} to up-regulate the expression of IFN-{alpha} gene and protein production and suggest that priming the production of endogenous IFN-{alpha} is a critical step in the mechanism of action of rhIFN-{alpha} in HCL.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HCL3 is a chronic lymphoproliferative disorder characterized by splenomegaly, pancytopenia, and the presence of typical HCs with long cytoplasmic processes in peripheral blood and bone marrow. Since the first report (1) , IFN-{alpha} has become an important therapeutic agent in HCL and represents an excellent example of successful cancer biotherapy (2) . Several mechanisms of action of IFN-{alpha} have been discussed, including a direct antiproliferative effect on the HCs (3) , blocking of autocrine growth factor loops (4) , activation of natural killer cells (5) , induction of terminal differentiation (6) , and modulation of the immunophenotype of HCs (7) . However, there is no agreement on which of these mechanisms is the determinant factor of the therapeutic effect of this cytokine (8) .

We have recently shown that, in patients with HCL, the in vitro production of hematopoietic growth factors is inadequate and, as demonstrated with interleukin 6, rhIFN-{alpha} is able to up-regulate its production (9) . It has also been suggested that the production of IFN-{alpha} is impaired in HCL as a consequence of monocytopenia (10) . The clinical relevance for such a deficiency was provided by demonstrating an inverse correlation between disease activity and the production of IFN-{alpha} by PBMCs on virus stimulation in vitro (11) . Nevertheless, there is no direct information on IFN-{alpha} gene expression in HCL, and virus stimulation in vitro may not precisely reflect how IFN-{alpha} gene is regulated in vivo in patients with HCL.

In healthy individuals, IFN-{alpha} mRNA is constitutively expressed in peripheral blood leukocytes and other organs such as spleen, kidney, and liver (12 , 13) . The synthesis of IFN-{alpha} is usually subjected to a stringent control, and significant quantities are produced on stimulation with inducers such as viruses or synthetic dsRNA (14) , whereby monocytes seem to be the major producer cells (15) . There is a growing evidence, however, that IFN-{alpha} production is not restricted to the monocytes; B and T lymphocytes as well as polymorphonuclear cells constitutively express IFN-{alpha} mRNA and produce IFN-{alpha} (16) . In addition, natural IFN-producing cells (NIPC) are generating substantial amounts of IFN-{alpha} in response to viral stimulation (17) . Several investigators, using sensitive immunoassays, have also found that plasma of healthy individuals may contain detectable amounts of IFN-{alpha} or IFN-{alpha}-like substance (18 , 19) . Under pathological conditions such as lymphocytic leukemia, Hodgkin’s disease, and bladder cancer, IFN-{alpha} production by PBMCs was found to be reduced (20, 21, 22, 23) , and low levels of circulating IFN-{alpha} have been reported in adult T-cell leukemia (24) .

To identify the pattern of IFN-{alpha} gene expression in HCL in vivo and to understand the mechanisms underlying its regulation, we measured the expression of IFN-{alpha} mRNA in freshly isolated PBMCs under basal conditions and on induction with rhIFN-{alpha} or dsRNA [poly(I·C)]. In a combined approach, we determined the levels of IFN-{alpha} in plasma of HCL patients and studied the intracellular levels and the capacity of the PBMCs to produce this cytokine. The results point to both impaired expression of IFN-{alpha} mRNA and low levels of circulating IFN-{alpha} in HCL patients. They also demonstrate that rhIFN-{alpha} is able to induce the expression of its own gene and to enhance production of endogenous IFN-{alpha}, thereby shedding light on new aspects in the mechanism of action of IFN-{alpha} in HCL.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
Ten patients with HCL and eight HDs were enrolled in the study, and samples were collected after informed consent. At the time of investigation, six patients were untreated and four patients were under therapy with rhIFN-{alpha} administered by s.c. injection of 2 x 106 units, three times a week. The diagnosis of HCL was based on the presence of the typical HCs in the peripheral blood and bone marrow as well as double immunofluorescence staining of the HCs using monoclonal antibodies against CD19 and CD11c. Clinical and hematological data are shown in Table 1Citation .


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  		Table 1 Hematological data of patients with HCL at time of investigation

 
Cell Cultures.
PBMCs were isolated from heparinized blood using Ficoll-Hypaque (Pharmacia Biotech Inc., Uppsala, Sweden) centrifugation. Fresh samples were either immediately subjected to RNA isolation (within 1–2 h of blood withdrawal) or incubated for 24 h in RPMI 1640 supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (all reagents were obtained from Life Technologies, Inc., Paisley, United Kingdom). Cells were cultured at a density of 2 x 106/ml either uninduced or induced with 100 IU/ml rhIFN-{alpha} (IFN-{alpha}2b was obtained from Schering-Plough, Kenilworth, NJ) or 100 µg/ml poly(I·C) (Pharmacia). After incubation for 24 h at 37°C in 5% CO2, cells were harvested, culture supernatants were collected for IFN-{alpha} protein assays, and cells were washed in PBS and processed for RNA isolation. In another set of experiments, cells were cultured in serum-free medium or in medium containing increasing concentrations (2.5, 5, 10, and 20%) of FCS, HD serum, or HCL serum and then were processed for mRNA analysis.

IFN-{alpha} Determination by RT-PCR.
RNA from identical cell numbers was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction techniques (25) using RNAzol B (TEL-TEST, Friendswood, TX). Integrity of RNA was controlled by gel electrophoresis and, subsequently, RNA was quantified spectrophotometrically. cDNA was synthesized from 1 µg of RNA. The synthesis efficiency in all of the samples was verified by 30 cycles of PCR using human ß-actin-specific primers (upstream, 5'-GAG CTG CGT GTG GCT CCC GAGG-3'; downstream, 5'-CGC AGG ATG GCA TGG CAT GGG GGA GGG CAT ACC CC-3'; Ref. 9 ). RT-PCR of IFN-{alpha} mRNA was performed using IFN-{alpha}-specific primers (upstream, 5'-AGA ATC TCT CIT TIC TCC TGI ITG AIG GAC AGA-3'; downstream, 5'- GAT CTC ATG ATT TCT GCT CTG ACA AC-3'; fragment size of 385 bp; Ref. 26 ). Forty cycles of amplification were performed (denaturation at 94°C for 1 min, annealing at 64°C for 1.5 min, and extension at 72°C for 1.5 min.). Amplified DNA was electrophoresed, stained with ethidium bromide, and photographed. The expression of IFN-{alpha} mRNA was corrected to ß-actin mRNA level in each sample.

Detection of IFN-{alpha} by Immunoassays.
PBMCs (2 x 106 cells/ml) were incubated with or without rhIFN-{alpha} or poly(I·C) as described above. Culture supernatants were collected and stored frozen at -80°C until the time of the assays. To insure that the detected amounts of IFN-{alpha} in the cultures treated with rhIFN-{alpha} were indeed attributable to production of endogenous IFN-{alpha}, parallel experiments were performed measuring the amounts of IFN-{alpha} recovered from cell-free medium supplemented with 100 IU/ml of rhIFN-{alpha} at the corresponding times of incubation. These values were subtracted from the detected concentrations of IFN-{alpha} in the test samples. Taking into account that the incubation procedures might have an effect on cell proliferation, cell cycle analysis and cell counting were performed at the end of the incubation periods. Standard ELISA kits that recognize IFN-{alpha}2 (Endogene Inc., Cambridge, MA) were used in all of the experiments and samples were measured in duplicate. The standards in these kits are calibrated to the NIAID standard lot Ga23-901-532 (1 pg = 1.68 NIAID units). Because IFN-{alpha} may be found in a bound form in the circulation (27 , 28) , we applied a new competitive EIA to measure the concentrations of IFN-{alpha} in plasma samples. The assay is designed to detect both free and bound forms of IFN-{alpha} in biological fluids (IFN-{alpha} Accucyte, CytImmune Inc., College Park, MD; 29 ).

Flow Cytometric Analysis.
PBMCs were washed in ice-cold PBS, fixed in 4% p-formaldehyde for 20 min at room temperature, and permeabilized by treatment with 0.05% NP40 (BDH Chemicals Ltd, Poole, England) in PBS for 5 min; they were then washed in PBS. Cells were stained with R-phycoerythrin-conjugated mouse monoclonal antihuman {alpha}-2-IFN antibodies (Chromaprobe Inc., Mountain View, CA) for 30 min. To identify the percentage of IFN-{alpha} producing cells within monocyte, B- and T-cell populations, double immunofluorescence staining was performed using IFN-{alpha} antibodies and FITC-conjugated mouse antihuman monoclonal antibodies that recognize CD14, CD19, and CD3 (all of which were obtained from Serotec, Kidlington, United Kingdom). Samples were analyzed by FACScan (Becton Dickinson, San Jose, CA). Twenty thousand events/sample were acquired with 4-decade logarithmic amplification using Lysys II software. In each sample, the respective isotype controls were used as a negative control to set the gates for data analysis. In a separate set of experiments, the absolute numbers of lymphocytes and monocytes per µl of blood were calculated from the percentages of these population within the WBC count provided in the clinical data of each individual patient. The absolute number of IFN-{alpha} producing cells within monocytes, B cells, and T cells in the peripheral blood was then calculated according to the percentage of IFN-{alpha} positive cells within these populations as measured by double fluorescence staining and FACS analysis.

Cell Enrichment.
To identify the cellular subpopulations contributing to the production of IFN-{alpha} in HCL, enrichment experiments were performed using MACS (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) with anti-CD19 or anti-CD14 antibodies for B cells and monocytes, respectively. T cells were obtained by negative selection. Enriched cell populations were further characterized by flow cytometry. The procedure resulted in cell enrichment of >97% of CD19+ B cells (B cells/HCs), >83% of CD14+ cells (monocytes), and >85% of CD3+ (T cells). Cells were cultured with and without poly(I·C) or rhIFN-{alpha}. FACS analysis was performed to determine the percentage of IFN-{alpha}-positive cells, and supernatants were collected for the evaluation of secreted IFN-{alpha} protein.

Statistical Analysis.
The results were analyzed for statistical significance by ANOVA, and P < 0.05 was considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{alpha} mRNA Expression.
As illustrated in Fig. 1ACitation , IFN-{alpha} mRNA was readily detected in freshly isolated PBMCs from HDs (Lanes 1–8). In contrast, under the same assay conditions, IFN-{alpha} mRNA was not detectable or only barely so in PBMCs from HCL patients (Lanes 9–18). The relatively weak signals found in three cases (Lanes 10–12) were obtained from patients under therapy with rhIFN-{alpha} (patients 2–4 in Table 1Citation ). Incubation of the cells for 24 h in medium (RPMI + 10% FCS) resulted in only a moderate enhancement of IFN-{alpha} mRNA in some of the HCL samples (Fig. 1B)Citation . When cells were treated with poly(I·C), an increase in IFN-{alpha} mRNA levels was observed in samples from HD as well as HCL patients, with the exception of one patient (Fig. 1CCitation , Lane 16; Table 1Citation , patient 8). Similarly, treatment with rhIFN-{alpha} (Fig. 1D)Citation , resulted in an increase of IFN-{alpha} mRNA in HD and HCL patients, again with the exception of the same patient. It is important to note that this particular patient had developed resistance to IFN-{alpha} therapy.



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  		Fig. 1. Expression of IFN-{alpha} mRNA in PBMCs of HDs (HD, Lanes 1–8) and HCL patients (HCL, Lanes 9–18). A, freshly isolated PBMCs; B, after incubation for 24 h in RPMI medium; C, after induction with poly(I·C); D, after incubation with rhIFN-{alpha}. ß-actin mRNA served as a control to ensure equal sample loading (A–D, lower tracks). Lanes 10–12 correspond to PBMCs from patients under IFN-{alpha} therapy.

 
The results of RT-PCR analysis were quantitated by scanning densitometry. The mean value of the integrated absorbance corresponding to the levels of IFN-{alpha} mRNA was significantly lower in the fresh samples from HCL patients than from HDs (0.018 versus 0.687; P < 0.001). Integrated absorbance corresponding to samples cultured in medium alone were also lower in HCL patients as compared with HDs (0.395 versus 0.654). Treatment with poly(I·C) or with rhIFN-{alpha} resulted in a remarkable increase in the levels of IFN-{alpha} mRNA, particularly in HCL patients. In HDs, the integrated absorbance values on induction with poly(I·C) and rhIFN-{alpha} were 2.709 and 2.777, respectively, and in HCL patients, 2.440 and 2.50, respectively. This indicates that rhIFN-{alpha} may enhance the expression of IFN-{alpha} mRNA in responsive patients to a level that is comparable with healthy individuals.

Effect of HCL Serum on IFN-{alpha} mRNA Expression.
To investigate the possibility that an inhibitory factor(s) for IFN-{alpha} mRNA expression might be present in the serum of HCL patients, PBMCs from a patient with HCL in remission under therapy with rhIFN-{alpha} (Table 1Citation , patient 2; Fig. 1Citation , Lane 10) were investigated. At this time point, PBMCs constitutively expressed IFN-{alpha} mRNA. Cells were cultured in medium containing 2.5, 5, 10, and 20% autologous serum that had been collected before initiation of therapy. A control experiment was run in parallel using HD serum or FCS. As demonstrated in Fig. 2Citation , no significant variation in IFN-{alpha} mRNA expression was observed in cells cultured in serum-free medium or in medium containing FCS or HD serum. However, in cultures supplemented with 10 and 20% of autologous HCL serum, inhibition in IFN-{alpha} mRNA expression occurred after 24 h. Similar results were observed when sera of other untreated patients were used and were consistent with this finding. The results point to the presence of a factor(s) in serum of untreated patients that might be responsible for the suppressed expression of IFN-{alpha} mRNA.



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  		Fig. 2. Effect of HCL serum on IFN-{alpha} mRNA expression (RT-PCR analysis). PBMCs from a patient in remission under IFN therapy were cultured for 6 h or 24 h in serum-free medium or in medium supplemented with different concentrations (2.5, 5, 10, or 20%) of either FCS, HD serum, or autologous serum obtained before therapy.

 
IFN-{alpha} Production by PBMCs.
Because the synthesis of IFN-{alpha} might be also regulated at the posttranscriptional level (30) , we investigated the effect of rhIFN-{alpha} or poly(I·C) on the production of IFN-{alpha} protein. Cells were cultured with or without these substances, and supernatants were tested for the presence of IFN-{alpha} by the standard ELISA technique. As demonstrated in Fig. 3Citation , no IFN-{alpha} was detectable in culture supernatants of HCL samples, whereas minimal amounts could be detected in HD samples. Incubation of the cells with poly(I·C) or rhIFN-{alpha} resulted in an increased production of IFN-{alpha}. However, the amounts of IFN-{alpha} in HCL patients remained significantly lower than in HDs. In HDs, the mean value on induction with IFN-{alpha} was 332 units/ml and with poly(I·C was 272 units/ml. In HCL patients, the corresponding values were 80 units/ml and 72 units/ml, respectively (P < 0.001). Cell counting and cell cycle analysis confirmed that the increase in IFN-{alpha} production was not attributable to an enhanced cell proliferation or variation in cell number (not shown).



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  		Fig. 3. IFN-{alpha} production by PBMCs from HCL patients and HDs. PBMCs obtained from 10 HCL patients and 8 HDs were cultured for 24 h. Culture supernatants were collected, and IFN-{alpha} was measured by immunoassays. Values, the mean concentration of IFN-{alpha} (units/ml) ± SD; +pIC, treated with poly(I·C); +IFN-{alpha}, treated with recombinant human IFN-{alpha}.

 
It is noteworthy that a correlation between the IFN-{alpha} mRNA expression and protein production was found, particularly in patients responding to rhIFN-{alpha} therapy. It is also important that PBMCs of a patient who was resistant to rhIFN-{alpha} (patient 8, Table 1Citation ; Fig. 1Citation , Lane 16), neither expressed IFN-{alpha} mRNA nor produced IFN-{alpha} protein and did not respond to in vitro induction.

Kinetics of IFN-{alpha} Production.
Kinetic studies on the production of IFN-{alpha} protein revealed that PBMCs from HCL patients respond to induction with rhIFN-{alpha} only after a prolonged lag phase. PBMCs from HDs (n = 3) produced IFN-{alpha} within 9 h after stimulation (mean, 28.5 units/ml) and showed a substantial increase after 24–48 h (mean, 245.6 and 245 units/ml, respectively), whereas PBMCs from HCL patients (n = 3) started to produce small amounts only after 12 h (mean, 11.2 units/ml) and showed a moderate increase after 24–48 h (means, 51.3 and 57.8 units/ml, respectively) but did not reach the values obtained with PBMCs of HDs.

Intracellular IFN-{alpha}.
The fact that the amounts of IFN-{alpha} protein produced by PBMCs were lower in HCL patients than in HDs may be attributable to both/either an impaired gene expression (as already demonstrated) and/or a deficiency of IFN-{alpha} producing cells. Therefore, freshly isolated PBMCs from the untreated patients (n = 4), from patients in remission under rhIFN-{alpha} therapy (n = 2), and from HDs (n = 4) were processed for FACS analysis to determine the percentage of IFN-{alpha}-positive cells within PBMCs, monocytes, B cells, and T-cell populations. In untreated patients, a severe reduction in the percentage of IFN-{alpha}-positive cells was found within all of the cell populations (P < 0.05), whereas the percentage of IFN-{alpha}-expressing cells in patients treated with rhIFN-{alpha} was comparable with that of HDs. A representative experiment is demonstrated in Fig. 4Citation . To get further insight into the in vivo situation in which pancytopenia is predominant in HCL patients, the numbers of IFN-{alpha}-positive cells per µl blood were calculated. As shown in Table 2Citation , as compared with HDs, a clear reduction in the absolute numbers of IFN-{alpha} positive cells within PBMCs, monocytes (CD14+), B cells (CD19+), and T cells (CD3+) was found in the untreated patients (patients 1, 6, and 7), whereas higher numbers of IFN-{alpha} positive-cells were seen in patients who were under therapy at the time of investigation (patients 2 and 4). It is important to note that in these two particular patients, the number of IFN-{alpha}-positive cells within monocytes, B cells, and T cells (not shown) were about three times lower before the initiation of therapy with rhIFN-{alpha}. This indicated that in untreated patients, not only the monocytes but also the B and T cells were deficient in producing IFN-{alpha} and that these cell populations may respond to induction by rhIFN-{alpha} in vivo. However, the conversion of the HCL cells into IFN-{alpha}-positive cells remained to be clarified.



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  		Fig. 4. FACS analysis of intracellular IFN-{alpha}. Staining was performed using antihuman phycoerythrin (PE)-conjugated IFN-{alpha} alone or in combination with either CD14, CD19, or CD3 antibodies. Values in the left upper quadratics, the percentage of IFN-{alpha} positive cells; values in the right upper quadratics, the double positive cells. A representative experiment is shown. HCL1, untreated patients; HCL2, patients under IFN-{alpha} therapy.

 

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  		Table 2 Absolute numbers of IFN-{alpha}-positive cells per µl of blood

Shown are the values calculated after flow cytometric analysis for the percentage of IFN-{alpha} positive cells within the PBMC fraction after Ficoll-Hypaque gradient centrifugation.

 
IFN-{alpha} Production by B Cells.
To identify the cell population that contributes to the defective production of IFN-{alpha}, PBMCs enriched for CD19+ cells from an untreated patient (92%, CD19+/CD11c+), from a patient under therapy with rhIFN-{alpha} (2%, CD19+/CD11c+), and from a HD were cultured either with or without poly(I·C) or rhIFN-{alpha}. As shown in Fig. 5Citation , the production of IFN-{alpha} by the B cells/HCs of the untreated patient was severely reduced as compared with the B cells of the treated patient and of the HD. Incubation with poly(I·C) or with rhIFN-{alpha} resulted in a slight increase in IFN-{alpha} production in the untreated patient, whereas a considerable increase was observed in the HD and in the treated patient. These data may indicate that the malignant cells (CD19+/CD11c+) of HCL patients are deficient in producing IFN-{alpha} as compared with their normal counterpart B cells. The presumption that HCs might be deficient in producing IFN-{alpha} was substantiated by immunohistochemical staining of intracellular IFN-{alpha}, which revealed a negative, or very weak, staining in the HCs as compared with normal B cells (not shown).



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  		Fig. 5. IFN-{alpha} secretion by B cells. MACS sorting technique was applied for enrichment of the B cells (>98% B cells) from an untreated patient (HCL1, >92% HCs), a patient under IFN therapy (HCL2, 3% HCs), and from a HD. Shown are the concentrations of IFN-{alpha} measured in supernatants by ELISA under basal conditions (Co) and after 24 h incubation with poly(I·C) (poly.I:C) or rhIFN-{alpha} (IFN-{alpha}).

 
IFN-{alpha} Levels in Plasma.
To find out whether expression of IFN-{alpha} mRNA may also reflect the in vivo response to IFN-{alpha} therapy, we measured the levels of circulating IFN-{alpha} in the plasma of treated and untreated HCL patients and of HDs. Because the cytokine might be present in a protein-bound form in biological fluids (27 , 28) , we applied a new competitive EIA, which is designed to detect free and bound IFN-{alpha} molecules. The results were compared with that obtained by the conventional sandwich ELISA technique. Using the latter method, we were not able to detect IFN-{alpha} in any of the plasma samples in HCL patients and in HDs. With the EIA method (Fig. 6)Citation , significant amounts of IFN-{alpha} were found in the plasma of HDs as well as in that of patients during the course of IFN-{alpha} therapy (patients 2–4, Table 1Citation , in whom PBMCs spontaneously express IFN-{alpha} mRNA; Fig. 1Citation , Lanes 10–12). No, or very low, levels were detected in the plasma of untreated patients (plasma which did not spontaneously express IFN-{alpha} mRNA (Fig. 1Citation , Lanes 9, and 13–18). These results confirm that up-regulation of IFN-{alpha} mRNA, in response to rhIFN-{alpha} therapy, is associated with increased concentrations of circulating IFN-{alpha}. The available assays do not directly distinguish between endogenously produced and exogenously administered IFN-{alpha}. However, because blood samples were withdrawn from the patients before s.c. injection of rhIFN-{alpha} (2–3 times a week) and on the basis of the pharmacokinetic profiles of injected IFN-{alpha}—where elimination half-life was found to be about 5 h and IFN-{alpha} was undetectable after 24 h of s.c. administration (31) —it is possible to speculate that the detected IFN-{alpha} in our study was the endogenously produced, rather than the exogenously administered, cytokine.



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  		Fig. 6. Levels of IFN-{alpha} in plasma. EIAs were used to measure circulating IFN-{alpha} in HDs (HD; n = 8), untreated patients (HCL + IFN-{alpha}; n = 6), and patients under rhIFN-{alpha} therapy (HCL; n = 4). In the treated patients, samples were collected before s.c. injection of rhIFN-{alpha}. No IFN-{alpha} was detectable by conventional ELISA technique.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study reveal a defective regulation of IFN-{alpha} gene at the transcriptional level in HCL and demonstrate an impaired supply of endogenous IFN-{alpha} by monocytes and B- and T-cell populations. They also demonstrate that IFN-{alpha} induces the expression of its own genes in vitro and in vivo, thereby presenting a novel mechanism of action of rhIFN-{alpha} in HCL and perhaps in other pathological conditions.

Freshly isolated PBMCs from untreated HCL patients, in contrast to HDs, did not express IFN-{alpha} mRNA. However, stimulation with rhIFN-{alpha} or with poly(I·C) resulted in a significant up-regulation of IFN-{alpha} mRNA, and this was associated with an enhanced production of IFN-{alpha} protein. To the best of our knowledge, an impaired expression of IFN-{alpha} mRNA in HCL has not been reported before. Several causes may have been responsible for this defect: (a) IFN-{alpha} mRNA may have been expressed at extremely low levels that could not be detected by RT-PCR conditions applied for the samples from HDs; (b) IFN-{alpha} may have been present as a subtype that differs from that in HDs and could not be detected by the primer pair used; and (c) IFN-{alpha} genes in PBMCs from HCL patients are under continuous inhibition by hitherto unknown factor(s). Indeed, there is evidence for the existence of an inhibitory activity in serum of HCL patients on normal progenitor cells as demonstrated by colony-forming assays (32 , 33) . We were also able to show that exposure of PBMCs to the serum of untreated patients leads to down-regulation of IFN-{alpha} mRNA. The exact nature of the inhibitory activity on the IFN-{alpha} gene needs further characterization.

Incubation of PBMCs with recombinant IFN-{alpha} resulted in a remarkable up-regulation of IFN-{alpha} mRNA in HCL patients as well as in HDs. Although it has been reported that IFN-{alpha} is able to prime its own production on stimulation with viruses or with a virus simulator such as poly(I·C) (34) , in the present study, rhIFN-{alpha} was found to induce the expression of its own genes independently from additional inducers. Whereas a moderate enhancement was seen in HDs, a considerable increase was found in HCL patients. This indicates the presence of repressed, but rather intact, IFN-{alpha} genes in PBMCs from HCL patients and excludes a constitutional gene deletion as has been reported in acute lymphoblastic leukemia (35) . Induction of IFN-{alpha} mRNA expression by poly(I·C)in PBMCs of HCL patients further confirms the presence of intact IFN-{alpha} genes and indicates that the observed increase in IFN-{alpha} mRNA is attributable to activation of transcription rather than to reduced mRNA degradation (36) .

The mechanisms responsible for the up-regulation of IFN-{alpha} mRNA by rhIFN-{alpha} is not completely clear at present. However, it may be attributable to an indirect effect of rhIFN-{alpha} through inhibiting a putative repressor factor(s) produced by the HCs, or it may be attributable to a priming effect of IFN-{alpha} (37) similar to that observed in patients with chronic hepatitis B under IFN-{alpha} therapy (38) . It is noteworthy that freshly isolated cells from patients with HCL who were under IFN-{alpha} therapy at the time of investigation showed detectable signals of IFN-{alpha} mRNA. Thus, indicating that the priming effect of rhIFN-{alpha} may also take place in HCL during IFN therapy. On the other hand, the observation that PBMCs from one patient did not express IFN-{alpha} mRNA, neither under basal conditions nor after induction, could be of clinical relevance because this patient is resistant to IFN-{alpha} therapy. These results indicate that the in vitro response to induction may mirror in vivo responsiveness to rhIFN-{alpha} therapy.

Despite the up-regulation of IFN-{alpha} mRNA, the amounts of IFN-{alpha} protein produced by PBMCs from HCL patients were lower than in HDs. This may be attributable to the dilution of IFN-{alpha}-producing cells by HCs in the samples. Indeed, flow cytometric analysis revealed a significant reduction in the number of IFN-{alpha}-expressing cells in untreated HCL patients. The HCs, themselves, seem to be defective in producing IFN-{alpha} protein. This was confirmed by immunohistochemistry studies on purified HCs as compared with normal B cells (not shown) and is underlined by the kinetic studies that demonstrate that the small amounts of IFN-{alpha} protein produced in HCL are detectable only after a prolonged lag phase.

It has been previously suggested that the impaired production of IFN-{alpha} in HCL might be attributable to the reduced number of monocytes (10 , 39) However, this may not be the sole reason. For instance, monocytes have been shown not to be necessary for IFN-{alpha} production on poly(I·C)stimulation (40) and normal B and T cells are also capable of producing IFN-{alpha} (16) . In addition, response to IFN-{alpha} therapy may take place before correction of monocyte counts in HCL (10) . Therefore, it is conceivable that other cell populations are also defective in producing IFN-{alpha} in HCL. In support of this postulation is that B-cell populations of untreated HCL patients (consisting of >90% HCs) produced minute amounts of IFN-{alpha}, even after stimulation with poly(I·C) or with IFN-{alpha}. In addition, the percentage of T cells expressing IFN-{alpha} was lower than that in treated patients and in HDs. Interestingly, a remarkable increase in the percentage of IFN-{alpha} positive cells within the monocyte and B-cell and T-cell populations was found in two patients during the course of IFN therapy (Table 2Citation ; Fig. 4Citation ). Thus, the impaired production of IFN-{alpha} in HCL patients is likely attributable to suppressed expression of IFN-{alpha} mRNA and synthesis of IFN-{alpha} proteins in several cell types. The data may support the notion that rhIFN-{alpha} enhances the production of IFN-{alpha} in vivo not only in the monocytes but also in B- and T-cell populations.

Detection of circulating IFN-{alpha} in the plasma of treated patients may be of clinical significance. Using the conventional sandwich ELISA technique, we were not able to detect IFN-{alpha} in plasma samples from HDs and HCL patients. With a new competitive EIA, however, remarkable amounts of IFN-{alpha} were detected in HDs and in treated, but not in untreated, patients. This may be attributable to the presence of the cytokine in a complex form in plasma that could not be detected by the sandwich ELISA. In fact, there is growing evidence for the presence of circulating endogenous IFN-{alpha} in plasma under normal conditions that contribute to the host defense mechanisms (18 , 19 , 41) . Under pathological conditions such as adult T-cell leukemia, a significant reduction in the level of circulating IFN-{alpha} as compared with that in HDs has been reported (24) . This situation may be similar to the situation with HCL. Lack of constitutive expression of IFN-{alpha} mRNA in HCL patients may, therefore, contribute to a deficient production of circulating IFN-{alpha}, hence explaining the need for continuous treatment with low doses of rhIFN-{alpha} to maintain its therapeutic effect in HCL (8) .

Provided that a priming effect may also occur in vivo, induction of IFN-{alpha} mRNA with subsequent production of endogenous IFN-{alpha} may lead to a constant regulation of several physiological processes such as immune recognition, cellular differentiation, and hematopoiesis. Taken together, the results of this study indicate that the beneficial effect of rhIFN-{alpha} in HCL is not attributable merely to replacement of this cytokine (42) . The up-regulation of IFN-{alpha} mRNA expression by rhIFN-{alpha} and the induction of endogenous IFN-{alpha} production seem to be equally important steps leading to the therapeutic effect of rhIFN-{alpha}. This may also explain the remarkable response to rhIFN-{alpha} therapy, even at relatively low doses.


   ACKNOWLEDGMENTS
 
We thank Drs. Gerhard Gruber and Dan Tong, and Sonja Bouzari, for helpful advice and stimulating discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the Commission of Oncology "Max-Kellner-Stipendium," Medical Faculty, University of Vienna and from the Austrian National Bank (Project No. 5198/6). Back

2 To whom requests for reprints should be addressed, at University of Vienna, Clinic of Internal Medicine I, Department of Hematology and L. Boltzmann Institute for Cytokine Research, Waehringer Guertel 18–20, A-1090, Vienna, Austria. Phone: 43-1-40400-5459; Fax: 43-1-40400-4461; E-mail: Josef.Schwarzmeier{at}akh-wien.ac.at Back

3 The abbreviations used are: HCL, HC leukemia; HC, hairy cell; rhIFN, recombinant human IFN; PBMC, peripheral blood mononuclear cell; dsRNA, double-stranded RNA; HD, healthy donor; RT-PCR, reverse transcription-PCR; EIA, enzyme immunoassay; FACS, fluorescence-activated cell sorting; MACS, magnetic cell-separation system; poly(I·C), polyionosinic-polycytidylic acid. Back

Received 2/ 1/00. Accepted 8/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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