Interleukin-13 levels in serum from patients with Hodgkin disease and healthy volunteers

The malignant Hodgkin and Reed-Sternberg (H/RS) cells of Hodgkin disease (HD) and the surrounding infiltrating cells are known to secrete several types of cytokines and chemokines, many of which have been implicated in the different clinical and histological presentations of HD.1-4 Most recently, interleukin-13 (IL-13) and IL-13 receptors have been shown to be expressed by cultured HD-derived cell lines and primary H/RS cells.5,6Furthermore, IL-13 has been detected in the supernatants of cultured HD-derived cell lines, and neutralizing antibody to IL-13 has been shown to inhibit the growth of these cell lines in vitro,5suggesting that H/RS cells may enhance their own survival by an IL-13 autocrine and paracrine cytokine loop.5 Because of the potential implication of these observations for treatment of HD, we examined IL-13 levels in serum samples from patients with newly diagnosed and relapsed HD and compared these levels with serum IL-13 levels in healthy volunteers.

IL-13 levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit from Biosource International (Camarillo, CA) and were measured in duplicate using a μ-Quant plate reader equipped with KC4 software (Biotech Instruments, Winooski, VT). The sensitivity of this ELISA is less than 12 pg/mL. All experiments included a set of standard wells containing known quantities of recombinant human IL-13. Results are reported as the average value of duplicate measurements. Supernatants from 3 HD-derived cell lines that are known to secrete IL-13 (HD-LM-2, L-428, and KMH2) were used as positive controls.5 Supernatants from one additional HD-derived cell line (HD-MYZ) were used for comparison. The cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (Department of Human and Animal Cell Cultures, Braunschweig, Germany).7 Cell lines were cultured (5 x 10⁵/mL) in RPMI medium supplemented with penicillin (5%), streptomycin (5%), and heat-inactivated fetal calf serum (10%). Supernatants were collected after 24 hours in culture and immediately assayed for IL-13 levels by ELISA. Serum from 108 consecutive patients with newly diagnosed HD and from 31 patients with relapsed HD was studied. Serum from 40 healthy donors was used for comparison. All serum samples were obtained after proper consent was granted and were stored in a freezer at −70°C until used.

Consistent with a previously published report, the 3 IL-13–producing cell lines that we examined expressed a range of IL-13 levels, from 85 to 300 pg/mL5 (Figure 1). The HD-MYZ cells did not secrete IL-13. None of the 40 serum samples that were obtained from healthy donors contained detectable levels of IL-13 (Figure 1). Subsequently, sera from 108 patients with newly diagnosed HD were examined. Of these patients, 70% had nodular sclerosis histology, and 36% had stage III or IV disease (Table1). Thirty-one patients (28%) had B symptoms. Eleven (10%) of the 108 serum samples contained detectable levels of IL-13 (Figure 1). In all 11 cases, IL-13 levels were at least 30 pg/mL (range, 34 to 82 pg/mL). The 3 patients with the highest IL-13 levels had nodular sclerosis histology; 2 had stage IIA disease, and one had stage IIB disease. IL-13 levels did not correlate with gender, disease stage, histological subtype, disease bulk, or presence of extranodal involvement. This lack of correlation may simply be due to the small number of patients who were found to have elevated serum IL-13 levels. There was a trend for a higher percentage of patients with B symptoms to have elevated IL-13 levels (45.8% vs 26.8%), but this difference was not statistically significant (χ²test). Of the 11 newly diagnosed patients who had elevated levels of IL-13, only one patient experienced disease progression, 4 months after completing therapy for stage IIB bulky mixed-cellularity disease. Interestingly, 2 of 12 patients with lymphocyte-predominant HD had elevated serum IL-13 levels. These data are in contrast with a previously published report that showed that IL-13 mRNA expression was limited to primary H/RS cells of the classic type.6 The source of IL-13 in the 2 patients with lymphocyte-predominant HD is not known (tissue blocks are not available to perform in situ hybridization of IL-13 mRNA), although the possibility of IL-13 being produced by the malignant cells cannot be ruled out.

We also studied IL-13 levels in serum samples from 31 patients with relapsed HD. All samples were collected at the time of active disease and before therapy was started. In this group, 28 patients had nodular sclerosis histology, and 3 had unclassified histologies. Five (16%) of 31 patients had elevated IL-13 levels; 3 had stage II disease, and 2 had stage III disease. Only one had B symptoms. The median serum IL-13 level was 46 pg/mL (range, 42 to 48 pg/mL).

Although the number of patients with elevated IL-13 levels was small, this does not rule out the possibility that the majority of the patients may have had clinically significant IL-13 levels at the site of disease. Our data confirm the potential clinical relevance of IL-13 in patients with HD and warrant further exploration of novel therapeutic strategies to examine the clinical significance of the IL-13 survival loop in patients with HD.