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Abstract

Objective

This study aimed to determine the changes in peripheral blood TBNK lymphocyte subsets in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and their relationship with the pathogenesis of AECOPD.

Methods

A cross-sectional study of 1252 hospitalized patients in Zhejiang Hospital was conducted. There were 162 patients in the AECOPD group and 1090 in the non-chronic obstructive pulmonary disease (COPD) group. The proportions of peripheral blood T helper cells, cytotoxic T cells, total B cells, total natural killer (NK) cells, and total T cells in the two groups were determined, and the CD4/CD8 ratio was calculated.

Results

The proportions of men and total natural killer cells, and the mean age were significantly higher in the AECOPD group than in the non-COPD group. The T helper cell, total T cell, and CD4/CD8 ratios were significantly decreased in the AECOPD group. A multivariate logistic regression analysis showed that male sex, age, the total T cell ratio, and the CD4/CD8 ratio were significantly associated with the incidence of AECOPD.

Conclusion

Cellular immune dysfunction in patients with AECOPD causes a decrease in total T lymphocytes and the CD4/CD8 ratio, which may be involved in the pathogenesis of AECOPD.

Keywords

Chronic obstructive pulmonary disease T lymphocyte subset natural killer cell B lymphocyte CD4/CD8 ratio immune dysfunction

Introduction

Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable chronic airway disease characterized by persistent airflow limitation and corresponding respiratory symptoms, but its pathogenesis is unknown. The pathological changes in COPD are predominantly airway and/or alveolar abnormalities, which are often associated with considerable exposure to noxious particles or gases.¹ According to the latest World Health Organization statistics, approximately 3 million people die of COPD every year worldwide, accounting for the fourth leading cause of death in the world.² In recent years, studies have shown that the occurrence and progression of COPD are closely related to the function of the immune system, and abnormal expression of peripheral blood T lymphocyte subsets in patients with AECOPD has become a research hotspot.^3,4 However, there have been few studies on the immune function of B lymphocytes and natural killer (NK) cells.

We compared sex, age, and the distribution of T, B, and NK (TBNK) lymphocytes between three groups of patients with AECOPD, those with respiratory infectious disease (RID), and those with respiratory non-infectious disease (RNID), and also compared patients with AECOPD and those without COPD. We examined the associations between influencing factors and the pathogenesis of AECOPD to analyze the changes in immune function in patients with AECOPD, and provide a basis for further research on the causes of AECOPD.

Materials and methods

Patients

We performed a cross-sectional study on 1483 patients who were hospitalized in the Department of Respiratory Medicine of Zhejiang Hospital from January 2019 to November 2021 and had been tested for TBNK lymphocyte subsets. The exclusion criteria were as follows: (1) patients with repeated hospitalization and repeated testing; (2) patients with malignant tumors; (3) patients with pulmonary fungal infection such as cryptococcus and aspergillus; and (4) patients with sepsis. Figure 1 shows the procedure of selecting the patients. The diagnosis of AECOPD complied with the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease: The GOLD Science Committee Report 2019.² The diagnosis of RID and RNID was obtained from case data. Patients’ blood samples were collected within 24 hours of admission.

Figure 1.

Procedure of selecting the patients.

This study was approved by the Ethics Review Committee of Zhejiang Hospital, and an exemption approval letter was obtained to waive the patient’s informed consent (approval no.: 118K). All personal information had already been de-identified to protect the patients’ privacy. The reporting of this study conforms to the STROBE guidelines.⁵

Methods

A volume of 2 mL of peripheral fasting venous blood was obtained from all patients in the morning and placed into an EDTA anticoagulation tube. The blood samples were sent to the laboratory of Zhejiang Hospital for testing. CD45+CD3+CD4+CD8+, CD45 + CD19 + CD16 + CD56 + four-color fluorescent labeling flow cytometry was used to determine CD3+ cells. These CD3+ cells comprised total T lymphocytes, CD3+CD4+ lymphocytes, which are mainly helper T (Th) cells, and CD3+CD8+ lymphocytes, which are mainly cytotoxic T (Tc) cells. CD19+ cells comprised total B lymphocytes, and CD16+ CD56+ cells comprised total NK cells. The CD3+CD4+/CD3+CD8+ ratio, namely the CD4/CD8 ratio, was calculated. The FACSCanto flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and Becton Dickinson Multitest kit were used to identify these cells. The operation was performed in strict accordance with the manufacturer’s instructions.

Statistical analysis

Measurement data that conformed to a normal or approximately normal distribution are expressed as the mean ± standard deviation. Comparison between multiple groups was performed by one-way analysis of variance, and pairwise comparison between groups was performed by the Bonferroni method. Non-normally distributed measurement data are expressed as the median (P₂₅, P₇₅), count data are expressed by the rate (%), and the Kruskal–Wallis rank sum test was used for comparison between multiple groups. Univariate and multivariate logistic regression analyses were used to identify factors associated with the incidence of AECOPD. P < 0.05 indicates a statistically significant difference. SPSS 25.0 (IBM Corp., Armonk, NY, USA) statistical software was used to process the data.

Results

Patients

A total of 1252 patients were included in the study. These patients were divided into the AECOPD group (n = 162) and the non-COPD group (n = 1090). The patients in the non-COPD group were divided into the RID group (n = 831) and the RNID group (n = 259) according to whether there was respiratory infection.

Comparison of the AECOPD, RID, and RNID groups

The proportion of male sex (H value = 15.824, P < 0.001), age (H value = 124.135, P < 0.001), the Th cell ratio (F value = 17.185, P < 0.001), the total NK cell ratio (H value = 58.713, P < 0.001), the total T cell ratio (H value = 45.533, P < 0.001), and the CD4/CD8 ratio (H value = 7.466, P = 0.024) were significantly different among the groups. Specifically, there were significant differences in the distribution of male sex, age, the Th cell ratio, the total NK cell ratio, the total T cell ratio, and the CD4/CD8 ratio between the AECOPD group and the RID and RNID groups (all P < 0.05). However, there was no significant difference in these factors between the RID and RNID groups (Table 1).

Table 1.

Comparison of the distribution of influencing factors among three groups of patients.

Influencing Factor	AECOPD group (n = 162)	RID group (n = 831)	RNID group (n = 259)	H/F value	P value
Male sex, n (%)	116 (71.6)	455 (54.8)^a	151 (58.3)^a	15.824	<0.001
Age (years), median (P₂₅, P₇₅)	79 (72, 84)	63 (48, 76)^a	65 (54, 75)^a	124.135	<0.001
Th cell ratio (%), mean ± SD	35.9 ± 11.3	41.3 ± 10.3^a	40.2 ± 10.9^a	17.185	<0.001
Tc cell ratio (%), median (P₂₅, P₇₅)	24.4 (18.4, 30.4)	23.9 (18.7, 30.7)	24.7 (18.9, 31.4)	1.223	0.542
B cell ratio (%), median (P₂₅, P₇₅)	10.8 (6.8, 16.4)	12.1 (8.2, 15.8)	12.0 (7.7, 16.7)	2.623	0.269
NK cell ratio (%), median (P₂₅, P₇₅)	20.0 (12.3, 28.7)	12.4 (8.1, 18.9)^a	12.5 (8.1, 20.6)^a	51.683	<0.001
T cell ratio (%), median (P₂₅, P₇₅)	64.2 (55.4, 73.1)	71.4 (63.9, 77.6)^a	70.2 (63.6, 77.1)^a	45.533	<0.001
CD4/CD8 ratio, median (P₂₅, P₇₅)	1.50 (1.02, 2.18)	1.71 (1.20, 2.46)^a	1.67 (1.18, 2.20)	7.466	0.024

AECOPD, acute exacerbation of chronic obstructive pulmonary disease; RID, respiratory infectious disease; RNID, respiratory non-infectious disease; SD, standard deviation; NK, natural killer.

P < 0.05, compared with the AECOPD group.

Comparison of the AECOPD and non-COPD groups

We compared the distribution of each influencing factor between the AECOPD and non-COPD groups. We found that the proportion of male sex (Z value =−3.846, P < 0.001), age (Z value = −11.101, P < 0.001), and the total NK cell ratio (Z value = −7.132, P < 0.001) were significantly higher in the AECOPD group than in the non-COPD group. The Th cell ratio (t value = 5.688, P < 0.001), total T cell ratio (Z value = −6.710, P < 0.001), and CD4/CD8 ratio (Z value = −2.237, P = 0.025) were significantly lower in the AECOPD group than in the non-COPD group (Table 2).

Table 2.

Comparison of the distribution of each influencing factor between the AECOPD and non-COPD groups.

Influencing factor	AECOPD group (n = 162)	Non-COPD group (n = 1090)	Z/t value	P value
Male sex, n (%)	116 (71.6)	606 (55.6)	−3.846	<0.001
Age (years), median (P₂₅, P₇₅)	79 (72, 84)	64 (51, 76)	−11.101	<0.001
Th cell ratio (%), mean ± SD	35.9 ± 11.3	41.0 ± 10.5	5.688	<0.001
Tc ratio (%), median (_P25, P₇₅)	24.5 (18.4, 30.4)	24.0 (18.8, 30.9)	−0.629	0.529
B cell ratio (%), median (P₂₅, P₇₅)	10.7 (6.8, 16.4)	12.1 (8.1, 16.0)	−1.619	0.105
NK cell ratio (%), median (P₂₅, P₇₅)	20.0 (12.3, 28.7)	12.4 (8.1, 19.2)	−7.132	<0.001
T cell ratio (%), median (P₂₅, P₇₅)	64.2 (55.4, 73.1)	71.1 (63.8, 77.4)	−6.710	<0.001
CD4/CD8 ratio, median (P₂₅, P₇₅)	1.50 (1.02, 2.18)	1.70 (1.19, 2.37)	−2.237	0.025

AECOPD, acute exacerbation of chronic obstructive pulmonary disease; COPD, chronic obstructive pulmonary disease; SD, standard deviation; NK, natural killer.

Regression analysis between various influencing factors and AECOPD

Univariate logistic regression was initially performed to examine the association between each influencing factor and the prevalence of AECOPD. AECOPD was the dependent variable, and sex, age, and the Th cell, Tc cell, total B cell, total NK cell, total T cell, and CD4/CD8 ratios were independent variables. We found that male sex (odds ratio [OR] = 2.014, 95% confidence interval [CI] 1.403–2.892, P < 0.001), age (OR = 1.075, 95% CI 1.060–1.091, P < 0.001), and the total NK ratio (OR = 1.054, 95% CI 1.040–1.069, P < 0.001) were positively associated with the incidence of AECOPD. The proportion of Th cells (OR = 0.957, 95% CI 0.942–0.972, P < 0.001) and the total proportion of T cells (OR = 0.952, 95% CI 0.939–0.965, P < 0.001) were negatively associated with the incidence of AECOPD (Table 3).

Table 3.

Univariate logistic regression analysis of each influencing factor and AECOPD.

Influencing factor	B value	OR value	95% CI	P value
Male sex (%)	0.700	2.014	1.403–2.892	<0.001
Age (years)	0.073	1.075	1.060–1.091	<0.001
Th cell ratio (%)	−0.044	0.957	0.942–0.972	<0.001
Tc cell ratio (%)	−0.006	0.994	0.977–1.010	0.450
B cell ratio (%)	−0.008	0.992	0.967–1.017	0.513
NK cell ratio (%)	0.053	1.054	1.040–1.069	<0.001
T cell ratio (%)	−0.049	0.952	0.939–0.965	<0.001
CD4/CD8 ratio	−0.112	0.894	0.762–1.050	0.171

OR, odds ratio; CI, confidence interval, NK, natural killer.

A multivariate logistic regression analysis was then performed. AECOPD was the dependent variable, and factors with P values <0.2 in the univariate logistic regression analysis, namely sex, age, and the Th cell, total NK cell, total T cell, and CD4/CD8 ratios, were included as independent variables into the regression equation. Male sex (OR = 1.666, 95% CI 1.133–2.450, P = 0.009), age (OR = 1.068, 95% CI 1.052–1.084, P < 0.001), the total T cell ratio (OR = 0.970, 95% CI 0.957–0.984, P < 0.001), and the CD4/CD8 ratio (OR = 0.852, 95% CI 0.727–0.998, P = 0.047) were significantly associated with the incidence of AECOPD (Table 4).

Table 4.

Multivariate logistic regression analysis of each influencing factor and AECOPD.

Influencing factor	B value	OR value	95% CI	P value
Male sex (%)	0.511	1.666	1.133–2.450	0.009
Age (years)	0.066	1.068	1.052–1.084	<0.001
T cell ratio (%)	−0.030	0.970	0.957–0.984	<0.001
CD4/CD8 ratio	−0.160	0.852	0.727–0.998	0.047

OR, odds ratio; CI, confidence interval.

Discussion

This study showed that the incidence of AECOPD was associated with male sex and age. The proportion of male sex and age in the AECOPD group were significantly higher than those in the non-COPD group. These results are consistent with those of epidemiological investigations of COPD in China and in other countries.^6–8

The relationship between COPD and human autoimmune function has attracted an increasing amount of attention in recent years. Lymphocytes belong to a class of white blood cells with specific immune functions, mainly including T lymphocytes involved in cellular immunity, B lymphocytes involved in humoral immunity, and NK cells that can directly kill target cells. T lymphocytes can be divided into Th cells (CD3+CD4+ T lymphocytes) and Tc cells (CD3+CD8+ T lymphocytes) according to their functions. The maintenance of normal immune function in the body depends on maintaining an appropriate proportion of TBNK cell subsets. This study showed that the peripheral blood Th cell, total T cell, and CD4/CD8 cell ratios were significantly lower, but the total NK cell ratio was higher, in the AECOPD group than the RID and RNID groups. However, there was no significant difference in the above-mentioned factors between the RID and RNID groups. These findings are in agreement with those of Zhou et al.⁹ Our findings suggest that immune dysfunction is involved in the pathogenesis and progression of AECOPD, which manifests as an abnormal distribution of TBNK subsets in peripheral blood.

The logistic regression analysis showed that sex, age, the total T lymphocyte ratio, and the CD4/CD8 ratio were significantly associated with the incidence of AECOPD. The total T cell lymphocyte and CD4/CD8 ratios in the AECOPD group were lower than those in the non-COPD group, which is consistent with the results of previous studies.^9,10 Although some studies have shown that changes in Tc and Th cells are related to the progression of AECOPD,¹¹ similar results were not obtained in this study. The reasons for this discrepancy between studies are as follows. T lymphocytes are mainly divided into Th and Tc cells according to the expression of CD4+ or CD8+. There are also regulatory/inhibitory T cells, memory T cells, and possibly even more as yet undiscovered subpopulations, which may affect Th and/or Tc cell ratios. However, the trend of a decrease in the Th cell ratio and an increase in the Tc cell ratio in the AECOPD group is consistent with these previous studies. The CD4/CD8 ratio can reflect this trend more sensitively, which can also explain the importance of calculating and presenting the CD4/CD8 ratio in clinical TBNK items.

NK cells are important innate immune cells that can non-specifically lyse certain tumor and virus-invaded target cells, and can also kill certain target cells through antibody-dependent cell-mediated cytotoxicity. NK cells regulate the function of other immune cells by secreting cytokines, and play an important role in the body’s immune surveillance and early anti-infection immunity. This study showed that the proportion of NK cells in peripheral blood was significantly higher in the AECOPD group than in the RID group or RNID group, which is consistent with the results of Hsu et al¹², indicating that NK cells are involved in the progression of AECOPD. In aggravation of the patient’s disease, NK cells are gradually activated to exert their immune regulation and anti-infection effects. However, in the multivariate regression analysis, the proportion of NK cells in peripheral blood was not significantly related to the pathogenesis of AECOPD. A possible reason for this lack of finding is that, when total T lymphocytes (CD3+ cells) are decreased, CD3⁻ cells, such as NK cells, are relatively rather than absolutely increased. Therefore, the quantitative and functional changes in NK cells in the peripheral blood of patients with COPD needs to be further studied.

This was a cross-sectional study with certain limitations. The enrolled patients were hospitalized patients in the acute phase in our hospital, and there may have been selection bias. Therefore, prospective cohort studies are required to further verify our findings.

In summary, patients with COPD have immune dysfunction and decreased cellular immune function, which mainly manifest as a decrease in the proportions of total T lymphocytes, Th cells, and the CD4+/CD8+ ratio, and an increased trend in the proportion of Tc cells. These cellular changes may be one of the pathogeneses of AECOPD. In the comprehensive treatment of patients with COPD, rational regulation of immune function, especially improvement of cellular immune function, may be beneficial for delaying disease progression and improving the prognosis of this disease.

Supplemental Material

sj-pdf-1-imr-10.1177_03000605231182556 - Supplemental material for Changes in peripheral blood TBNK lymphocyte subsets and their association with acute exacerbation of chronic obstructive pulmonary disease

Supplemental material, sj-pdf-1-imr-10.1177_03000605231182556 for Changes in peripheral blood TBNK lymphocyte subsets and their association with acute exacerbation of chronic obstructive pulmonary disease by Xiufang Hong and Zhen Xiao in Journal of International Medical Research

Supplemental Material

sj-pdf-2-imr-10.1177_03000605231182556 - Supplemental material for Changes in peripheral blood TBNK lymphocyte subsets and their association with acute exacerbation of chronic obstructive pulmonary disease

Supplemental material, sj-pdf-2-imr-10.1177_03000605231182556 for Changes in peripheral blood TBNK lymphocyte subsets and their association with acute exacerbation of chronic obstructive pulmonary disease by Xiufang Hong and Zhen Xiao in Journal of International Medical Research

Footnotes

Author contributions

Xiufang Hong designed the experiments, analyzed the data, and wrote the manuscript. Zhen Xiao collected and analyzed the data, and revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Data availability statement

Data are available on request from the authors.

Declaration of conflicting interests

The authors declare that there is no conflict of interest.

Funding

Xiufang Hong was supported by funds from the Science and Technology Department of Zhejiang Province (2018KY198).

ORCID iDs

Xiufang Hong

Zhen Xiao

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