Eosinophilia and Cellular Cytokine Responsiveness in Hemodialysis Patients1’2
Joachim Hertel, Paul L. Kimmel,3 Terence M. Phillips, and Juan P. Bosch
uniform. Eosinophilia is a clinically useful marker of J. Hertel, P.L. Kimmel, TM. Phillips, J.P. Bosch, Depart- exaggerated HD-associated cytokine production. ment of Medicine, George Washington University Med- Cytokine production depends on individual respon- ical Center. Washington. D.C. siveness and is probably related to atopy. Cytokines
(J. Am. Soc. Nephrol. 1992; 3:1244-1252) may play an important role in the pathogenesis of HD-associated eosinophilia and hypersensitivity.
Key Words: lnferleukin- 1, interleukin-2, cuprammonium ABSTRACT H emodialysis (HD)-induced leukopenia and easin- Eosinophilia in hemodialysis (HD) patients has been aphilia have long been recognized, but their associated with allergy to dialyzers and exagger- clinical significance has been uncertain ( 1 ,2). Cam- ated activation of complement during HD. Its etiol- plement activation has been associated with many ogy, however, remains unknown. Complement adverse effects, such as heukopenia. hypatensian, activation can lead to cytokine production, and hypoxemia, pruritus, hypersensitivity reactions, and interleukin-2 (lL-2) administration has been shown to edema (3-6). The etiology and significance of HD- cause eosinophilia. Because abnormalities in cellu- associated easinaphilia, however, remains unclear. lar cytokine production in renal patients were previ- Recent biacampatibility studies have focused an the production of cytokines, peptide regulators re- ously demonstrated, the relationship between di- leased from a variety of cells, including activated alysis-associated eosinophilia and IL production in leukacytes. The most well-studied cytokmnes are in- this HD population was studied. Twelve patients on terleukin-1 (IL-i) and interleukin-2 (IL-2). The nor- chronic HD therapy with normal eosinophil counts mal kidney has important regulatory and excretory (mean, 0.23 ± 0.03 cells/nL) were compared with functions for many hormones and small-molecular- nine patients with eosinophilia (mean, 0.85 ± 0.17 weight proteins such as cytokines (pohypeptides sized cells/nL). Measurements of cellular IL-I and lL-2 pro- between 10,000 and 25,000 d) (7). Ninety percent of duction were performed before (pre) and after (post) administered IL- 1 was excreted by the kidney In a HD with cuprammonium dialyzers. In patients with murine model (8). The half-life of IL-2 greatly in- eosinophilia, stimulated cellular IL-I production in- creases after ligation of the renal arteries. IL-2 may be metabolized by the renal tubule (9,10). creased by I 17 ± 40% (P < 0.01) when post-HD Cytokines are important mediators of the immune measurements were compared with pre-HD values response. Their activation has been proposed as an and IL-2 production increased by 127 ± 65% (P < explanation for both acute and chronic complications 0.05). In contrast, there was no difference in stimu- in patients with ESRD treated with HD (1 1-13). Cir- lated cellular cytokine production when values be- culating levels and cellular production of bath cyta- fore and after HD were compared in patients without kines are generally, though not consistently, reported eosinophilia. Individual responses were reproducible to be increased in uremia and ESRD (14-20). The during subsequent dialysis. It was concluded that role of HD in the abnormal control of cytakine regu- cellular cytokine production in response to HD is not hation is not clear, because patients with chronic renal failure, before ESRD therapy is initiated, have evidence of abnormal stimulated cellular IL- 1 and IL- 2 production (18). I Received June 15, 1992. Accepted September 8. 1992. Easinaphilia has been reported after the adminis- 2 These data were presented in part at the American Federation for clinical tration of recombinant IL-2 (2 1 -24) and is also cam- Research, Baltimore, MD, May 1992. manly found in HD patients (25-28). Because HD 3 correspondence to Dr. P.L. Kimmel, Division of Renal Diseases and Hyperten- sion, Department of Medicine, George Washington University Medical center, Induces cytokmne production including IL-2 (15,18), 2150 Pennsylvania Avenue, NW, Room 4-425. Washington, D.C. 20037. we studied the relationship between cellular cytokine 1046-6673/0306- 1244$03.00/0 production and dialysis-associated easinaphilla in Journal of the American Society of Nephrology copyright C 1992 by the American Society of Nephrology our patients.
I 244 Volume 3 . Number 6 . 1992 Hertel et al
METHODS mented with 1 0% autalogaus serum before the total cell volume was divided into two batches. The cells Patients were incubated at 37#{176}C,and the media were replen- Twenty one patients with ESRD ranging in age ished before one batch was pulsed with 10 g,/mh of from 35 to 7 1 yr (mean, 56.9 ± 2.6 yr) were studied. lipapahysaccharide for the measurement of IL- 1 and Fifteen were male, and 6 were female; 20 were black, the other was pulsed with 50 tg/mL of phytohemag- and 1 was white. The underlying disease was chronic glutinin far the measurement of IL-2 far a further 24 glamerulonephritis In nine, hypertension in seven, h at 37#{176}C.After this second incubation, the cells diabetes mellitus in three, and chronic interstitial were centrifuged and the supernatants were used in nephrltls in two. All patients were treated with the stimulation assay. One hundred micrahiters of thrice-weekly chronic maintenance HD for 1 to 166 each supernatant was serially diluted and incubated months (mean, 51.4 ± 10.3 months). All were he- with either the CRL or CTLL target cell cultures for madialyzed with cuprammanlum dialyzers (5N or 6N: 48 h at 37#{176}C.The target cells were centrifuged, re- Gambra, Lund, Sweden) with an acetate bath. Five plenished with fresh media, and pulsed with tritiated milliliters of whole blood was drawn before (pre) and thymidine for 24 h at 37#{176}C.The target cells were after (post) a 3- to 4-h HD treatment at the time of harvested, and the amount of incorporated radloac- routine monthly laboratory testing. All patients gave tivity was measured in a Beckman LS 1 308 scintIlla- their Informed consent. The study was approved by tion counter (Beckman Instruments, Palo Alto, CA). the George Washington University Medical Center Data are expressed as tritium incorporation In counts Committee on Human Research. per minute (cpm) per culture. Intracellular IL-2 levels were also measured by an Eosinophil Counts antigen capture ELISA (34) wIth an anti-human IL-2 Eoslnophlh counts were measured with an auto- antibody solid-phase manoclanal antibody (35) (Ad- mated cell cytometer (H- 1 System: Technicon, Tar- vanced Biatechnohogles, Columbia, MD) as both the rytown, NY). The easinophil counts are expressed in solid-phase capture reagent and the reporter agent. cells per nanohiter. Eoslnophil counts were measured The ELISA was also used to measure intracellular IL- concomitantly with the cytokmne measurements at 2 levels after sonic disruption and ultracentrifuga- the time of monthly routine laboratory evaluation in tion clarification of the supernatant. The samples 1 7 of 2 1 patients. The presence of eoslnophihla was were compared with a standard curve, constructed defined as 0.38 cehls/nL or more. A normal eosinophil by incubating the target cells with known amounts count was defined as 0.37 celhs/nL or fewer. This of human IL-2 (Advanced Blatechnolagles). Data are cut-off has been found useful previously in patients expressed as tritium incorporation in counts per mm- with ESRD (29). PatIents with an underlying disease ute and nanagrams of IL-2 per milliliter as measured associated with eoslnophllla (such as drug reactions by ELISA. or parasitic diseases) were excluded. Data were analyzed by t test comparing differences In order to assure that the study group with easin- between pre-HD and post-HD treatment values ophihla and the control group without easinaphilia (paired t test) and comparing patients with easino- were different, six monthly easinophil counts preced- phihia and those without (unpaired t test). Cytokmne ing the measurement of IL were reviewed. Patients levels and easmnophil counts determined at the same with both persistent and intermittent eosinophilia HD treatment were used in regression analyses. Car- were Included In the eosinophllia group, if averaged relations were assessed by simple regression and eoslnophll counts from the patients’ three highest of were analyzed by the coefficient of variance. For the preceding six monthly values were more than calculations when the magnitude of cytokmne produc- 0.38 cells/nL. tion was mare Important than the absolute values, the differences (deltas [i]) between past-HD and pre- Cytokine Assays HD values were assessed. Alpha <0.05 was chosen as the level of significance. Data are expressed as As previously described (18), cellular IL-i and IL-2 mean ± standard error. production was measured by bioassay by the method of Clemens, Morris, and Gearing (30) with CRL 1445 IL- 1 -dependent fibroblasts (3 1) and CTLL IL-2- RESULTS dependent lymphocytes (32), respectIvely, as target Eosinophilia cells. Briefly, whale blood was collected in citrated glass tubes, mononuclear cells were separated by The mean eosinaphil count in all study subjects density centrifugatlon on Ficall-Hypaque (Pharma- was 0.49 ± 0. 1 0 cehhs/nL (range, 0.07 to 1 .7 1 cehhs/ cia, Plscataway, NJ), and their viability was checked nL). Twelve patients had normal easinaphil counts by trypan blue exclusion (33). The cell suspension with a mean of 0.23 ± 0.03 cells/nL (range. 0.07 to was adjusted to 5 X 1 06 cehls/mL In medium supple- 0.37 cehhs/nL). Their age was 55.4 ± 3.3 yr, and they
Journal of the American Society of Nephrology I 245 Eoslnophllia and Cellular Cyfokine Responsiveness
1) (13,753 ± 3,912 cpm) than patients without eosln- had been treated with HD for 40.8 ± 8.8 months. Nine patients had eosinophllIa, with a mean eoslno- ophihla (602 ± 1.071 cpm) (P < 0.01). phil count of 0.85 ± 0. 1 7 cells/nL (range. 0.38 to 1 .7 1 cells/nL). These patients were 58.9 ± 4.4 yr of IL-2 age and had been treated with HD for 65.4 ± 20.9 Stimulated cellular IL-2 production In patients with months. There was no significant difference between and wIthout easinophilia, before and after HD, Is patients with or without eosinophllla In terms of race, shown In Figure 2. Patients without eosinophIhla had age, sex, underlying disease. age, or time on HD. mean phytohemagglutinin-stimulated IL-2 measure- ments of 1 0,053 ± 389 cpm before dialysis (range, IL-I 8,000 to 1 2,63 1 cpm). Patients with eoslnophllla had pre-HD IL-2 values of 1 2,278 ± 1 ,962 cpm (range, Stimulated cellular IL- 1 production In patients with 5,886 to 26, 1 90 cpm). There was no difference In and without eoslnophllla, before and after HD, Is mean pre-IL-2 values between the two groups. Pa- shown In Figure 1 . Patients without eoslnophilla be- tients without easlnophilia had a mean post-HD IL-2 fore HD (pre-HD) had mean llpopolysaccharlde-stlm- value of 10,796 ± 1 ,063 cpm (range, 5,667 to 18,066 ulated IL-i levels of 10,716 ± 1 .031 cpm (range, cpm). The 7.2 ± 8.8% Increase compared with pre- 7,006 to 20,51 1 cpm). Patients with eosinophllia had HD values was not significant. Patients with easino- pre-HD IL- 1 values of 1 3,37 1 ± 2,849 cpm (range, philla had a mean post-HD IL-2 value of 24,735 ± 5,90 1 to 32. 1 96 cpm). There was no difference be- 5,840 cpm (range, 6,2 1 1 to 53,855 cpm)-an in- tween pre-IL- 1 levels In the two groups. After HD crease of 127.2 ± 65.2% compared with pre-HD val- (post-HD), mean stimulated cellular IL- 1 productIon ues (P < 0.05). Patients with eoslnophllia had a In patients without eoslnophlhla was 1 1 ,3 1 9 ± 1,212 greater dIfference between mean pre-HD and post- cpm (range, 5,799 to 2 1 .333 cpm). The 9.2 ± 9.9% HD values of IL-2 ( IL-2) (12,457 ± 5,218 cpm) than increase compared with mean pre-HD values was not patients without eoslnophllla (743 ± 957 cpm) (P < signIficant. In contrast, patients with eosinophllia 0.02). had mean post-HD IL- 1 values of 27, 1 23 ± 5,430 cpm (range, 8,063 to 47,035 cpm)-an increase of IL-2 by ELISA 1 16.7 ± 39.7% compared with pre-HD values (P < 0.0 1). PatIents with eoslnophllla had a greater differ- IL-2 bioassay measurements were confirmed by ence between pre-HD and post-HD values of IL- 1 ( IL- ELISA. The correlation between the two methods was
80000 60000
50000 50000
40000 40000 E E a’ a.
.E 30000 .E 30000 N
20000 20000 ‘S
10000 10000
0 0 IIIII pre post pro post pro post pre post
Eos neg Eos pos Eos neg Eos pos
FIgure 1. Measurements of stimulated cellular IL-I produc- Figure 2. Measurements of stimulated cellular 11-2 produc- tlon before (pre) and after (post) HD, comparing patients tion before (pre) and after (post) HD, comparing patients without eosinophilia (Eos neg; N = 12) and patients with without eosinophilia (Eos neg; N = 12) and patients with eosinophllla (Eos p05; N = 9). eosinophilia (Eos pos; N = 9).
1246 Volume 3 ‘ Number 6 ‘ 1992 . Hertel et al
0.98 (P < 0.001). Patients without easinaphihla had Correlation Between IL-I and lL-2 mean pre-HD IL-2 values of 32.5 ± 2.2 ng/mL (range, Measurements 23 to 45 ng/mL.). Patients with easinophihla had pre- Because IL- 1 productIon by macrophages may af- HD IL-2 values of 39.0 ± 6.4 (range, 14 to 75 ng/mL). feet IL-i production by lymphocytes, the relationship There was no difference between mean pre-HD val- between the two cytokines was also analyzed by car- ues In the two groups. After HD, patients without relating paired stimulated cellular IL- 1 and IL-2 pro- eoslnophlhla had mean post-HD IL-2 measurements duction levels. Stimulated cellular IL- 1 and IL-2 pro- of 37.3 ± 5.3 ng/mL (range, 14 to 74 ng/mL). The duction correlated before (r = 0.70; P < 0.001) and increase of 1 2.5 ± 1 0.6% compared with mean pre- after HD (r = 0.9 1 : P < 0.00 i ). The difference be- HD values was not significant. Patients with easino- tween cellular IL- 1 and IL-2 production was carre- phihla had a mean post-HD IL-2 value of 83.6 ± 19.7 hated as well (r = 0.89: P < 0.00 i ). This correlation ngJmL (range, 1 8 to 1 64 ng/mL)-an increase of between ML- 1 and XIL-2 was sIgnificant when pa- 178.0 ± 104.9% compared with pre-HD values (P < tients without easmnaphilia (r = 0.77: P < 0.01) or 0.05). PatIents wIth eosinophihla had significantly with easinaphihia (r = 0.86: P < 0.01) were analyzed greater post-HD IL-2 values (P < 0.02) and differences separately. The slopes and the intercepts of the between mean pre-HD and post-HD (ML-2) values of regression lines were not different when patients stimulated cellular IL-2 production by ELISA (44.6 ± with and without easmnaphihia were compared (Figure 19.0) than did patients without easinophlhia (4.8 ± 5). 4. 1 ng/mL) (P < 0.05).
Correlation Between Cytokine Measurements and Eosinophil Counts There was no correlation between individual paired E absolute eoslnophil counts and cytakine values be- a. fore HD In the 1 7 patients who had simultaneous .E determinations. Post-HD IL- 1 values, however, car- related with eoslnophil counts (r = 0.60: P < 0.02). There was no significant correlation of IL-2 and ea- sinophil counts after HD (r = 0.37: P 0. 1 5). Easin- ophil counts and IL- 1 values correlated with the dif- ference between post-HD and pre-HD levels ( \) (r = 0.74; P < 0.001) (Figure 3). but the correlation be- tween eosinophll counts and ML-2 values did not 0.2 0.4 0.6 0.8 1.0 1.2 1.4 reach significance (r = 0.44; P 0.08) (FIgure 4). Eosinophil Count (in cells/nL)
Figure 4. Correlation between concomitant eosinophil counts and the difference between post-HD and pre-HD r=0.74 350tE A measurements (. ) for stimulated cellular 11-2 production (N= 17).