Increased number and activation of peripheral basophils in adult‐onset minimal change disease

Abstract Nowadays, the pathogenesis of minimal change disease (MCD) is still not well‐known, and the current understanding on MCD is mainly based on data derived from children, and very few adults. Here, we comprehensively analysed the correlation between the changes of peripheral basophils and the incidence rate and relapse of adult‐onset MCD. The results showed that in patients at the onset of MCD, the ratio and activation of basophils were all higher than those of healthy controls (all P < .05). In vitro test results showed that basophils from healthy controls can be activated by the serum taken from patients with MCD. Among 62 patients at the onset of MCD, with complete remission after treatment and 1 year of follow‐up, the relative and absolute basophil counts before treatment were higher in the long‐term remission group (n = 33) than that of the relapse group (n = 29). The basophil counts were significantly higher in the infrequent relapse group (n = 13) than that of the frequent relapse group (n = 16; P < .05). These findings suggested that basophil may play a pathogenic role in adult‐onset MCD, and the increased number and activation of peripheral basophils could predict recurrence in adult MCD.

In vitro test results showed that basophils from healthy controls can be activated by the serum taken from patients with MCD. Among 62 patients at the onset of MCD, with complete remission after treatment and 1 year of follow-up, the relative and absolute basophil counts before treatment were higher in the long-term remission group (n = 33) than that of the relapse group (n = 29). The basophil counts were significantly higher in the infrequent relapse group (n = 13) than that of the frequent relapse group (n = 16; P < .05). These findings suggested that basophil may play a pathogenic role in adult-onset MCD, and the increased number and activation of peripheral basophils could predict recurrence in adult MCD.

K E Y W O R D S
activation, adult, basophil, minimal change disease, recurrence levels of IL-4 and IL-13 can participate in the onset of MCD. [7][8][9][10][11][12] Importantly, patients with MCD have low serum IgG levels 13 and elevated IgE levels, 14,15 suggesting that humoural immunity is involved in the onset of MCD. The onset of MCD is related to circulating immune factors 16

| Patients and controls
This study included patients at the initial onset of MCD, who were admitted to the Affiliated Hospital of Guangdong Medical University from January 2015 to February 2018. All patients were adults, and were diagnosed with idiopathic MCD, and met the diagnostic criteria of nephrotic syndrome. 21 The control groups were healthy physical examinees whose gender and age were matched with the experimental groups.

| Therapeutic regimen
Oral prednisone 1 mg/kg/d or an equal dose of methylprednisolone was used as an original dose. Then it was reduced by about 10% of the original dose every 2 weeks. Until the dose of prednisone was 10 mg/d, it was maintained for about 6 months and then the drug was slowly withdrawn. The total therapeutic course was 9-12 months. In patients with steroid resistance or frequent relapse, it might be combined with an immunosuppressant, such as cyclophosphamide.

| Definitions
Relapses were defined as follows: (a) relapse, relapse after urine protein was negative with urine protein ≥1+; (b) frequent relapse, more than two episodes of relapse in 6 months or more than three episodes of relapse in 1 year; (c) long-term remission, no recurrence after urine protein was negative for more than 1 year. 22,23

| Follow-up procedure in patients with MCD
Cases from 2015 to 2016 were retrospectively analysed for different relapse outcomes after the treatment and differences in the counts and proportions of basophils before treatment (as determined by routine blood tests). There were 85 patients at the initial onset of MCD, who had follow-ups until December 2017; 62 patients, who had follow-ups for 1 year after their urinary protein was negative after initial treatment; 33 patients with long-term remission; and 29 patients with relapse (including 16 cases of frequent relapse and 13 cases of infrequent relapse).

| Experimental method and flow cytometry analysis
Peripheral venous blood samples were collected from patients at the initial onset of MCD, beginning in 2017, who had received no glucocorticoids or immunosuppressants. The samples were processed within 2 hours, and flow cytometry was used to determine basophil (CD123 + CD203c + ) ratios, activation indexes (mean fluorescence in- A FACScanto™ II flow cytometer (Becton Dickinson) and the FlowJo 7.6 software were used to acquire and analyse the data, respectively.
In addition, number of basophils in peripheral blood of some patients with MCD were also determined by routine blood tests with automated numeration technics (Beckman coulter COULTER ® LH 750 System) for linear correlation analysis between FCM tests and routine blood tests.

| Effect of serum from patients with MCD on the activation of basophils
Blood was collected from healthy volunteers (n = 9) to negatively select basophils and separate the serum. Serum was then collected from nine patients with MCD. EasySep™ human basophil enrichment kit (Stemcell Technologies) was used to negatively purify human basophils to obtain a purity of greater than 90% using flow cytometry.
Nine portions of basophils from normal individuals were placed into RPMI 1640 medium (Gibco Laboratories) with serum from patients with MCD (20%) and IL-3 (20 ng/mL; PeproTech), then cultivated for 24 hours. Flow cytometry was then used to detect changes in the MFIs of CD203c and CD62L on basophils. Using the same method, nine portions of basophils from normal people were placed into the culture medium with allogeneic serum from healthy volunteers (20%) and IL-3 (20 ng/mL). Before FCM test, cell viability was tested by 0.2% trypan blue solution (Sigma).

| Ethical approval
The study was approved by the ethics committee of the Affiliated Hospital of Guangdong Medical University (approval no. PJ2017054).
The study conformed to the tenets of the Declaration of Helsinki. All patients provided informed consent before taking part in the study.

| Statistical analysis
All statistical analysis was performed using SPSS 23.0 (SPSS, Inc).
The normal distributed data were expressed as mean ± standard deviation, and two-group comparisons were performed using paired or unpaired two-tailed t test. Non-normal distributed data were expressed as median (P 25 , P 75 ), and two-group comparisons were performed using Mann-Whitney U test. Linear regression analysis was used to determine correlation between routine blood tests and flow cytometry tests of peripheral basophil counts. P < .05 was considered to indicate statistical significance.

| Patients at the initial onset of MCD had increased counts and enhanced activity of peripheral basophils
From March 2017 to February 2018, peripheral blood samples were collected from 17 patients at the initial onset of MCD (not yet treated) at our hospital. No statistical significance was found for age differences between patients and normal control groups (P > .05).
Flow cytometry analysis demonstrated that the mean ratio of peripheral basophils in white blood cells (0.48% ± 0.29%) in the MCD group was significantly higher than that of the normal control group (0.31% ± 0.17%) (P < .05; Figure 1).
During the same period under the same conditions, MFI values of CD203c and CD62L on basophils from patients with MCD were all higher than those of the normal control group (P < .05; Figure 2; Table 1).
Flow cytometry further detected the expression of intracellular factors of basophils before treatment in patients with MCD. The percentages of IL-4 + basophils and IL-6 + basophils to total basophils in the MCD group (n = 15) were all higher than those of the normal control group (n = 16; P < .05). The percentage of IL-13 + basophils was extremely low in the MCD group, and there was no significant difference compared with that of the normal control group (P > .05; Figure 3; Table 2).

| Serum from patients with MCD activated basophils from healthy controls
After basophils from healthy controls were cultivated and stimulated in the culture medium with an allogeneic serum of normal individuals for 24 hours, the cell viability was all higher than 95%, and the MFI values of activated and labelled CD203c and CD62L showed no significant differences (P > .05). After cultivation and stimulation in the culture medium with serum from patients with MCD, the MFI values of CD203c and CD62L were all significantly increased (P < .05; Table 3).

| Peripheral basophil counts assess MCD activity
Based on the above experimental findings, peripheral basophils from patients with MCD showed significantly increased quantity and enhanced activation. However, routine blood tests are used in clinical practice to obtain quantitative findings; therefore, we next used routine blood tests to evaluate the peripheral blood from the above-mentioned 16 patients with MCD. There were no significant differences in the ratios of basophils, as determined by paired t tests (P = .731). Linear correlation analysis indicated that the two methods (FCM and routine blood tests) were significantly correlated (r = .915, P < .001; Figure 4). The above results indicated that the two methods were consistent regarding the detection of changes in basophil counts.
Next, we used retrospective analyses to investigate whether basophil count alone could be used to assess MCD activity. First, differences in peripheral basophil counts were investigated between the active and remission phases in patients with MCD. There were 25 cases of patients at the initial onset of MCD admitted into our hospital from 2015 to 2016, who had complete remission after a full course of treatment (>9 months). These patients had stopped glucocorticoid treatment for more than 1 month (allowing the influence of glucocorticoids on white blood cell counts and basophil counts to be excluded), and routine blood tests were then performed again to determine the ratio and count of basophils. The basophil ratio during the active stage (initial onset, before treatment) (0.61% ± 0.34%) was notably higher than the remission stage (0.36% ± 0.17%) (P < .001; Figure 5A). The basophil count in the active stage (51.9 ± 31.3/μL) was significantly higher than the remission stage (31.0 ± 12.7/μL) (P < .01; Figure 5B). Therefore, the basophil ratio and count during the active stage were higher than those during the remission stage in patients with MCD. These results suggested that peripheral basophil counts assess MCD activity.

| Peripheral basophil counts could predict remission and relapse in patients with MCD
Another critical clinical issue is the peripheral basophil counts during remission and relapse in patients with MCD. There were 62 patients at the initial onset of MCD admitted from 2015 to 2016, who had negative urinary protein after the initial treatment and had follow-ups for 1 year, including 33 cases of long-term remission and 29 cases of relapse (16 cases of frequent relapse and 13 cases on infrequent relapse). The results indicated that the basophil ratio before treatment in the long-term remission group (0.61% ± 0.32%, n = 33) was higher than the relapse group (0.42% ± 0.24%, n = 29) (P < .05; Figure 6A).  The basophil count before the treatment in the long-term remission group (52.5 ± 33.0/μL, n = 33) was also higher than the relapse group (34.7 ± 17.0/μL, n = 29; P < .01; Figure 6B). The basophil ratio before the treatment in the infrequent relapse group (0.53% ± 0.25%, n = 13) was higher than the frequent relapse group (0.33% ± 0.20%, n = 16) (P < .05; Figure 6C). Notably, the basophil count before treatment in the infrequent relapse group (38.6 ± 14.1/μL) was not significantly different compared with the frequent relapse group (31.5 ± 18.8/μL) (P = .276). The basophil ratios in the long-term remission group, infrequent relapse group, and frequent relapse group showed gradual decreasing trends ( Figure 6D). These results suggested that patients at the initial onset of MCD with a relatively high basophil ratio and count are more likely to have long-term remission after treatment.

| D ISCUSS I ON
IgE and basophils maybe involved in the pathogenesis of MCD, 14,18 and  antibodies, which activate basophils to promote the pathogenic cycle. 25,26 The basophils in the peripheral blood white cells can be accurately detected using antibodies targeting CD123 and CD203c, two basophil-sensitive activation indicators. [27][28][29] Thus, activated basophils can directly affect the functions of T and B lymphocytes or indirectly affect T and B lymphocytes via generation of specific metabolites.
In our current study, we found that peripheral basophils in adult patients at the initial onset of MCD showed increased number and enhanced activity compared with that in patients with stable MCD.
These findings were consistent with the results described by Pirotzky et al, 18 further indicating that peripheral basophils in patients at the initial onset of MCD were activated and changes in the number and activation of basophils in patients with MCD were closely related to the activity of the disease.
Although the pathogenic factors associated with proteinuria in patients with MCD have not been defined, such pathogenic factors may originate from the blood circulation rather than the kidney. 16,30 When the serum of patients with MCD was used to treat basophils from healthy volunteers, basophils were activated, suggesting that the substance activating basophils may have originated from the serum, for example, IgE. The ratio of basophils positively expressing cytokines, including IL-4, IL-6, and IL-13, is another parameter for the activation of peripheral basophils in patients with MCD. Compared with CD203c and CD62L, this ratio puts more emphasis on the functional expression of activated basophils, that is, whether the activated basophils can secrete active mediators. Researchers have concluded that basophils produce the essential cytokine IL-4, which is required for the differentiation of Th2 cells, whereas IgE is involved in the main pathway mediating Th2-cell activation. 35 In our current study, we confirmed that the increased percentage of IL-4-positive basophils TA B L E 3 Changes in the activated parameters after basophils from normal individuals were stimulated and cultivated with serum from patients with MCD for 24 h

Activation index
Before stimulated (Normal, n = 9) After stimulated (Normal, n = 9) P After stimulated (MCD, n = 9) P  Certain inherent limitations were noted within the current study. One limitation of our study is that patients with non-MCD nephrotic syndrome were not included as a control group, so changes and activation of basophils might not necessarily be unique in patients with nephrotic syndrome. Another is, there maybe have the presence of CD123 + pDCs in basophil gate due to lower expression of CD203c by basophils from normal controls, which may lead the real data for basophil frequency of normal control may slightly lower. Also, the results showed that compared with MCD patients, the frequency of basophils was significantly lower than MCD, even there maybe have the presence of pDCs in basophil gate of normal control. So, this interference did not affect the conclusion of this study.
These findings suggested that basophil may play a pathogenic role in adult-onset MCD, and the increased number and activation of peripheral basophils could predict recurrence in adult MCD.

ACK N OWLED G EM ENTS
The study was supported by Natural Science Foundation of

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
HH, YX and QP provided the idea and conceived and designed the experiments. HH, SL, XC and XL performed the experiments and analysed the data. HH, HX, SW and CY wrote the manuscript. HL supervised the study. All authors reviewed and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data included in this study are available upon request by contact with the corresponding author.