Human β-Defensin 2 Concentration of Respiratory Tract Mucosa in Elderly Patients With Pneumonia and Its Associated Factors

ORIGINAL ARTICLE

Human b-defensin 2 Concentration of Respiratory Tract Mucosa in Elderly Patients with Pneumonia

and Its Associated Factors

Kuntjoro Harimurti*, Samsuridjal Djauzi*, Arief B. Witarto**, Esthika Dewiasty*

Department of Internal Medicine, Faculty of Medicine, University of Indonesia – dr. Cipto Mangunkusumo Hospital.

Jl. Diponegoro no. 71, Jakarta Pusat 10430, Indonesia. ** Laboratory of Biotechnology, Indonesian Institute of Science, Serpong, Banten, Indonesia.

Correspondence mail to: kuntjoro.harimurti01@ui.ac.id

ABSTRACT

Aim: to identify HBD2 peptide in sputum of patients with pneumonia; to obtain mean concentration difference of HBD2 between elderly patients and the younger adults with pneumonia; and to find any association between age, nutritional status, smoking habits, diabetes mellitus, and chronic obstructive pulmonary disease (COPD) and the concentration of HBD2 in patients with pneumonia.

Methods: a cross-sectional study with consecutive sampling technique was conducted in 23 elderly patients and 38 younger adults with pneumonia who were hospitalized in Cipto Mangunkusumo Hospital, Jakarta. Patients with pulmonary and respiratory tract malignancy, taking long- term corticosteroid and/or immunosuppressant therapy were excluded. The sputum of patient was taken spontaneously or by sputum induction technique and prepared for identification by dissolving with dithiothreitol (DTT) solution. The presence of HBD2 was identified by using SDS-PAGE and immunoblotting;

while the concentration was measured by ELISA. The mean difference of HBD2 concentrations between elderly patients and the young adults was analyzed using t-test. Chi-square test was performed to analyze the association between several risk factors and HBD2 concentrations in the sputum.

Results: the mean concentration of HBD2 in the sputum of all subjects was 178.98 (SD 49.55) pg/ml. There was no mean concentration difference of HBD2 between elderly and younger adult patients with pneumonia. Age, nutritional status, smoking habit and diabetes mellitus were not associated with HBD2 concentration; however, COPD was associated with HBD2 concentration (p-value = 0.014).

Conclusion: there is no mean concentration difference of HBD2 in the sputum of elderly and younger adult with pneumonia. There is association between COPD with HBD2 concentrations in the sputum of patients with pneumonia.

Key words: elderly, human β-defensin 2 (HBD2), ELISA.

INTRODUCTION

Pneumonia is still a great problem in the elderly population. In addition to its high incidence, it has a significant impact in this population, including high mortality rate, long hospitalization period, increased multidrug resistance to various causing microorganisms, as well as utilization of health care resources.^1-3 Altered immune function due to aging (immunesenescence), including the immune function of respiratory tract mucosa, is one of important factors that may increase the risk of pneumonia in elderly population. The immune function in respiratory tract mucosa, starting from the trachea to the alveolar system, play role on the development of respiratory and pulmonary infection such as pneumonia, since the first contact between microorganisms causing infection and their human hosts occurs here.⁴

Defensin is one of antimicrobial peptides in vertebrates, which is hypothesized to have very important role in the innate immune system (including immunity of respiratory tract mucosa) against bacterial infection, particularly Gram-negative bacteria, as well as virus infections.^5,6 Of various kinds of defensins that have been identified in human, the human β-defensin 2 (HBD2) is assumed to have important role in respiratory tract and pulmonary infection.^7-9 Thus, the identification of altered response of HBD2 concentration in respiratory tract mucosa and its risk factors in elderly population are essential, considering that the elderly are susceptible to respiratory tract and pulmonary infection and prone to have severe complication due to the infection.^2,3

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All ELISA procedures were run in room temperature (20-23^oC). The ELISA procedures include: reagent preparation, preparation of HBD2 standard, and ELISA HBD2 protocols according to manufacturer manual.¹⁰ Absorbance of optical density was read at 450 nm using Macrotiter Plate Reader. All measurements were done in duplo.

Statistical Analysis

Measurement of HBD2 concentration was presented as mean and its standard deviation. The mean difference of HBD2 concentrations between elderly and younger adult patients with pneumonia was tested by independent t-test. Multivariate analysis using logistic regression was done to identify risk factors that may associated with HBD2 concentration in the respiratory tract. Since there is no clear definition of normal HBD2 concentration in the case of infection, we used cut-off of mean HBD2 of all patients to differentiate between high and low HBD2 concentration. All hypothetical tests used significance level of <5% and 95% confidence interval were used when necessary.

RESULTS

Subject Characteristics

From September 2009 to April 2010, a total of 61 patients with pneumonia were recruited at the Emergency Room of Cipto Mangunkusumo Hospital, Jakarta, consisting of 23 (37.7%) elderly patients and 38 (62.3%) young adult patients. The mean age of all subjects in our study was 53.8 years (standard deviation [SD] of 16.34 years) and range of age between 14 and 83 years. The proportion of male subjects was 39.3%

of all subjects. The subject characteristics of our study based on elderly and young adult group are shown on Table 1.

Sample Preparation and SDS-PAGE Results Dissolved sputum samples of study subjects using dithiothreitol (DTT) solution and the abovementioned techniques indicated that the sputum filtrates were relatively homogenous in the low-viscosity solution (Figure 1).

Identification of HBD2 by SDS-PAGE technique revealed that in the DTT-prepared sputum samples, protein in the sputum could be separated according to their molecular weight (MW) although the separation was relatively vague (Figure 2). Human b-defensin 2 having an MW of 4.2 kD are on bottom side of the gel (arrow).

Our study was aimed to identify HBD2 in the respiratory tract mucosa of patients with pneumonia;

to obtain mean concentration difference of HBD2 in the sputum of elderly pneumonic patients and the young adults; and to find any association between various risk factors of diminished immunity and HBD2 concentration in the sputum of patients with pneumonia.

METHODS

Design, Setting, and Population

We conducted a cross-sectional study in patients with community-acquired pneumonia (CAP) at the Emergency Room of Cipto Mangunkusumo Hospital, Jakarta between September 2009 and April 2010. The exclusion criteria were patients who had pulmonary diseases other than CAP, including chronic bronchitis, chronic obstructive pulmonary disease, pulmonary tuberculosis, bronchiectasis, cystic fibrosis, pulmonary edema, and primary pulmonary malignancy, as well as patients with extra pulmonary diseases that may also affect the lungs and/or respiratory tract such as pulmonary metastasis of extra-pulmonary malignancy, and pulmonary edema due to heart failure or chronic kidney disease.

Sample Preparation, and Identification and Measurement of HBD2

Of all subjects, sputum specimens were obtained both spontaneously and through sputum induction.

Before further procedures and analysis, all sputum was refrigerated at -80⁰C. To isolate specific proteins in the sputum, we added dithiothreitol (DTT) with a ratio of 1:1 to the thawed sputum to reach relatively low viscosity sputum. Low viscosity sputum was needed for identification and measurements of specific proteins in the sputum using ELISA technique.

Preparation of sputum using DDT was never reported earlier and therefore it was necessary to be tested before measurement of HBD2 concentration in the prepared sputum. We used two techniques to identify HBD2 in the sputum before identification and measurement by ELISA, i.e.: by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and immunoblotting. With SDS-PAGE, molecular weight HBD2 of 4.2kD was identified by comparing isolated band in the gel with its standard.

Identification and measurement of HBD2 by ELISA was done using Human BD-2 ELISA Development Kit purchased from Phoenix Pharmaceuticals, Inc.

Kuntjoro Harimurti Acta Med Indones-Indones J Intern Med

Table 1. Subject characteristics Variables Elderly Group

(>60 years) N=23

Young Adult Group (18-60 years)

N=38 Age, mean (SD) 69.7 (6.93) 44.2 (12.49)

Sex male (%) 9 (39.1) 15 (39.4)

Education (%) - Uneducated/less

than elementary

school 4 (17.3) 8 (21.1)

- Elementary and

Junior High 11 (47.8) 17 (44.8)

- Senior High

School 3 (13.0) 12 (31.6)

- University 5 (21.7) 1 (2.6)

Occupation (%)

- Housewives 9 (39.1) 15 (39.5)

- Employee (governmental and

non-governmental) - 5 (13.1)

- Retired 8 (34.7) 1 (2.6)

- Entrepreneur 1 (4.3) 5 (13.2)

- Others 5 (21.7) 12 (31.6) Marital status (%)

- Married 12 (52.2) 33 (86.8)

- Widow/widower 11 (47.8) 1 (2.6)

- Single - 4 (10.5)

Smoking habit (%)

- Never 12 (52.2) 24 (63.2)

- Former 9 (39.1) 6 (15.8)

- Current 1 (4.3) 8 (21.1)

BMI, mean (SD) 22.7 (4.65) 21.3 (5.06) MNA score, mean

(SD) 18.9 (4.51) 17.26 (4.60)

Albumin level, mean

(SD) 3.48 (0.524) 3.34 (0.766)

Co-morbidity (%)

- Diabetes mellitus 8 (34.8) 8 (21.1)

- COPD 5 (21.7) 3 (7.9)

Abbreviations: SD=standard deviation, BMI=body mass index, MNA=mini nutritional assessment, COPD=chronic obstructive pulmonary disease

Figure 1. Sputum samples before (A) and after (B) been dissolved with dithiothreitol (DTT) solution

Figure 2. Result of protein separation according to their molecular weight (MW). Human β-defensin 2 proteins with MW of 4.2 kD are on bottom side of the gel in SDS-PAGE (arrow).

Concentration of HBD2 in The Sputum of Patients with Pneumonia and Its Association with Various Risk Factors

Forty six from 61 patients were available for measurement of HBD2 by ELISA technique. We found that the mean (SD) of HBD2 concentration in 46 patients with pneumonia was 178.98 (49.55) pg/ml.

There was no mean difference of HBD2 concentration between elderly patients (185.16 [52.66] pg/ml) and

young adult patients (174.24 [47.52] pg/ml). The mean difference of both groups was -10.93 pg/ml (95% confidence of interval (CI) -40.78 to 18.93 pg/

ml, p-value 0.47).

By categorizing HBD2 concentration into high and low level with cut off point on mean HBD2 concentration of all patients of 178.98 pg/ml, we found 20 patients with high HBD2 concentration and 26 patients with low HBD2 concentration. The chi- square test significantly associated between high HBD2 concentration and the presence of COPD. While age, nutritional status, smoking habits and diabetes mellitus were not associated with HBD2 concentration (Table 2).

Vol 43 • Number 4 • October 2011 Human b-defensin 2 Concentration of Respiratory Tract Mucosa in Elderly

patients with community-acquired pneumonia (CAP).

The WB technique was not sensitive enough to obtain HBD2 concentration for quantitative measurement.¹⁷

Until now, there has been no study especially evaluated the HBD2 concentrations either in the sputum, BAL fluid, or serum of elderly patients having infection such as pneumonia. A study that had specifically evaluated the difference of HBD2 expression between young adult and elderly subjects was conducted by Matsuzaka et al.¹⁸ The study which used immunostaining technique indicated that there was a different location of HBD2 expression in the gingival tissue of both groups. In the elderly groups, HBD2 expression was largely found in superficial layer of pancreatin gingival epithelium; while in the young adult subjects the expression of HBD2 was mostly found in the upper layer of spinosus cells of pancreatin gingival epithelium.¹⁸ The study had not determined HBD2 concentration in those subjects quantitatively.

In this study, we found no difference of HBD2 concentrations between elderly patients and younger adults with pneumonia. As a component of innate immunity, it is assumed the HBD2 production would be lower in elderly subjects compared to the young adults during acute infection. In addition to the induction by bacterial or other microorganism products, HBD2 production is also stimulated by pro-inflammatory cytokines, which are normally produced during infection or inflammation.¹⁹ Moreover, nuclear factor (NF)-κB as an element of innate immunity system is a key component in HBD2 production. Some studies demonstrated that the induction may also be facilitated by mitogen-activated protein kinase (MAPK) pathway.⁷ Those three components inducing HBD2 had been known to have reduced activity with increasing age.²⁰

There are some possibilities about why we could not find any difference in mean HBD2 concentrations between elderly patients and the young adults in our study. First, there was no difference in production or synthesis of HBD2 during acute respiratory tract infection between elderly and young adult patients.

Although there have been numerous evidence that the adaptive immune system is altered or diminished with increasing age, the altered innate immunity system, including altered soluble mediators of respiratory tract mucosa such as HBD2 in elderly patients is still controversial.²¹ Age itself is frequently not a determinant factor of altered immunity system in a subject. Other co-morbidities generally have more effects on immune deficiency condition.²² It may also occur in the subjects of our study. Most of the young

Table 2. Association between age, nutritional status, smoking status, diabetes mellitus, and COPD with HBD2 concentration

HBD2 Concentration High, n=20 Low, n=26 p value Age >60 years 9 (45.0%) 11 (42.5%) 0.855 Malnutrition 10 (50.0%) 13 (50.0%) 1.000

Smoking status 0.586

Never 13 (65.0%) 13 (50.0%)

Former 4 (20.0%) 8 (30.8%)

Current 3 (15.0%) 5 (19.2%)

Diabetes mellitus 13 (65.0%) 21 (80.8%) 0.227

COPD 19 (73.1%) 20 (100%) 0.014

DISCUSSION

Various studies analyzing a range of proteins and cells contained in the sputum expelled by patients indicated that the phase of processing sputum is quite crucial. Dithiothreitol (DTT) as the solvent or liquefier of further sputum processing has been studied widely along with its associated effect on proteins and other substance contained in the sputum. Recent studies about DTT utilization in sputum processing were conducted by Pignatti et al., Woolhouse et al.,and Erin et al. Those studies indicate that adding DTT for sputum processing would only slightly affect the number of cells and the concentration of various soluble mediators will be analyzed further.^11-13

The human b-defensin 2 (HBD2) concentration in pulmonary tissue and respiratory tract mucosa increases due to infection caused by bacteria, viruses, or fungi.^5,6,14 Studies on HBD2 concentration in the respiratory tract and lungs usually use bronchoalveolar lavage (BAL) or epithelial lining fluid (ELF) specimens since they have low viscosity which makes them easily analyzed by using the ELISA technique.

Patients with diffuse panbronchiolitis (DPB) had greater HBD2 concentration (264 [6.1] pg/ml), which had 3.8 times fold increase compared to normal subjects (69.2 [20.0] pg/ml, p=0.01).¹⁵ Using the Western Blot technique, Singh et al. demonstrated that HBD2 was not detected in the BAL specimens of healthy subjects;

however, it increased in patients with cystic fibrosis (ranged from 0.1 to 10 pg/ml) and it had further increase in patients with pulmonary inflammation (ranged from 10 to 100 pg/ml).¹⁶ Human BD2 detection in the sputum has not been frequently performed due to the high viscosity of the sputum. A study in patients with pneumonia had identified HBD2 antimicrobial peptide using Western Blot (WB) technique in the sputum of

Kuntjoro Harimurti Acta Med Indones-Indones J Intern Med

CONCLUSION

In this study we could identify an antimicrobial peptide, human β-defensin 2 (HBD2), in the sputum of patients with pneumonia. The mean HBD2 concentration in the sputum of patients with pneumonia is 178.98 pg/ml. However, there is no mean difference of HBD2 concentration between elderly and younger adult patient. There is an association between COPD co-morbidity and HBD2 concentration; while the age, nutritional status, smoking habit, and diabetes mellitus are not associated with HBD2 concentration in the sputum of patients with pneumonia.

ACKNOWLEDGEMENT

Authors would like to say thank you to Juliyanti, MD and Lydia Tantoso, MD for their valuable effort in collecting data for this study.

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adult patients with pneumonia in our study also had other co-morbidities that are likely to reduce immune function so that they easily contract pneumonia. Other co-morbidities include patients with chronic kidney disease, and systemic lupus erythematosus (SLE) who are taking immunosuppressant therapy, and patients with malnutrition.

The second possibility that may explain why there was no significant difference in HBD2 concentration is that elderly patients who had pneumonia and in need of hospitalization in general, have a more severe disease and more likely to have multiple etiologic microorganism compared to the young adults.²³ Severe infection and multiple microorganisms are known to be able to induce production/synthesis of HBD2 in the site of infection.¹⁶

We also found that the concentration of HBD2 was associated with the presence of COPD, while there was no association between HBD2 concentrations with age, nutritional status, smoking status, and diabetes mellitus.

Patients with COPD are known to be more susceptible to occurrence of respiratory tract and pulmonary infection. Antimicrobial peptides have been shown to display a variety of activities that may implicate them in the pathogenesis of COPD. This is based on the observation that they not only contribute to defense against respiratory pathogens that have been associated with COPD, but may also contribute to the influx of inflammatory cells, activation of adaptive immunity, and epithelial remodeling.²⁴

To our knowledge, this is the first study which aimed to identify and measure the presence of HBD2 in the sputum of elderly patients with pneumonia and compare it with younger adult patients. This is also the first study which tried to find any association between HBD2 concentrations with several risk factors of diminished immunity. The result of analyses mostly did not attain statistical significance; but we believe that the result can be used as a basis for future study about HBD2 (or innate immunity in general) in elderly population. This study also has several limitations.

First, we used cross-sectional design that could not establish any cause-effect relationship between several risk factors of diminished innate immunity and HBD2 concentrations. Second, since there is no clear definition of normal HBD2 concentration in the case of infection, the cut-off value that we used in this study may lead to measurement bias. But since the bias is undifferential (it is not associated with the presence of risk factors), this only has minor influence to the results and conclusions.

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