Article, Respiratory Medicine

Inhaled corticosteroids increase blood neutrophil count by decreasing the expression of neutrophil adhesion molecules Mac-1 and L-selectin

a b s t r a c t

Objective: The objective was to investigate the effect of commonly used inhaled corticosteroids on white blood cell count and to examine the mechanisms involved.

Methods: This randomized comparative study comprised 60 Healthy adults. We measured the effects of budesonide (by face mask inhalation or aerosol inhaler), fluticasone (by inhaler), and saline inhalation (control) on WBC and the differential leukocyte count, especially the absolute Neutrophil count (ANC). To elucidate the mechanisms involved, we measured the expression of the adhesion neutrophil ligands Mac-1 (CD11b) and L- selectin (CD62L), and granulocyte colony-stimulating factor serum levels.

Results: Six hours after a single-dose inhalation of budesonide, mean increases of 23.4% in WBC (95% confidence interval [CI], 11.3-35.4) and 30.1% in ANC (95% CI, 7.2-53.0) were noted. The percentage of neutrophils increased from 54.6% to 58.1% (Pb .001). Inhaled fluticasone increased WBC and ANC by 12.6% (95% CI, 1.5-23.7) and 22.7% (95% CI, 6.2-39.2), respectively (Pb .01 for both). The absolute lymphocyte and eosinophil counts did not change significantly from baseline.

The expression of Mac-1 and L-selectin decreased by 51.0% (Pb .01) and 30.9% (P= .02), respectively, following face mask inhalation of budesonide and by 39.8% (P= .01) and 17.4% (P= .17), respectively, following inhalation of fluticasone. No significant changes in granulocyte colony-stimulating factor levels were noted.

Conclusions: Glucocorticoid inhalation increases WBC by increasing ANC. Reduced neutrophil adhesion to the en- dothelial surface, mediated by decreased adhesion molecule expression on neutrophils, is a plausible mechanism. Physicians should be aware of the effect of inhaled corticosteroids on WBC, as it may influence Clinical decisions, especially in the emergency department.

(C) 2016

Introduction

Peripheral white blood cell count and especially the absolute neutrophil count (ANC) and the proportion of neutrophils of total WBC are markers of inflammation and infection that are commonly used as essential components in many clinical decisions [1-4]. These markers are often incorporated in algorithms guiding the management of patients in the emergency department (ED), for example, febrile young infants [5] and suspicion of meningitis [6] or bacteremia [7].

? This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

* Corresponding author at: Department of Pediatrics A, Schneider Children’s Medical Center, Petach Tikva 49202, Israel. Tel.: +972 39253137, +972 545256900 (Mobile);

fax: +972 39253958.

E-mail address: [email protected] (Y. Pasternak).

1 Co-first authors with equal contribution.

Systemic (oral or intravenous) administration of corticosteroids (CSs) has been shown to increase WBC and ANC in a dose-dependent manner [8-11]. At 4-6 hours after administration of systemic CSs, a peak effect on WBC and ANC was measured, with return to basal levels within 24 hours [9]. Several mechanisms were proposed to explain the pathophysiology of the systemic CS-induced elevations in WBC and ANC, including enhanced release of neutrophils from bone marrow, delayed apoptosis, reduced migration of neutrophils into tissues, and a shift of neutrophils from the marginating to the circulating pool [12-16]. In particular, reduction in the adhesion neutrophil ligands Mac-1(CD11b) and L-selectin (CD62L), which results in reduced adhe- sion of neutrophils to the endothelial surface, is a well-established mechanism of increased neutrophil count by systemic CS administra- tion [13,15].

To avoid the significant adverse effects associated with systemic CS administration, inhaled CSs are currently key components in the treat- ment of acute wheezing episodes and asthma, and also other respiratory diseases, and are thus increasingly used worldwide [17-21]. Very low

http://dx.doi.org/10.1016/j.ajem.2016.07.003 0735-6757/(C) 2016

systemic absorption of inhaled CSs has been documented, with minor effects on the hypothalamic-pituitary-adrenal axis, as well as on bone, carbohydrate, and lipid metabolism [19,20-23].

WBC and ANC are frequently used diagnostic tools in acutely ill pa- tients seeking medical attention for respiratory symptoms, especially in the ED [3]. However, a considerable portion of these patients are treated with inhaled CSs before arriving at the ED or during their stay there. Clarification of the effect of inhaled CS administration on WBC and ANC is important for the proper interpretation of the blood cell counts of these patients.

Few studies evaluated the effects of inhaled CSs on WBC and ANC, mostly for beclomethasone, in a small number of subjects and with equivocal results [20,24]. The present study aims to examine the effects of the currently most commonly used inhaled CSs, budesonide and fluticasone, on WBC and ANC and attempts to elucidate the mechanisms involved. By examining the effect of a single-dose inhalation of CS, the present study was designed to address this issue from the point of view of the physician in the ED.

Materials and methods

A randomized, controlled, 2-phase study was conducted. Approval by the Institutional Review Board for Human Studies was obtained.

Subjects

Healthy volunteers were recruited to the study. Inclusion criteria were age 18 to 50 years, healthy with no significant underlying disease, no use of systemic medication, no use of systemic or inhaled steroids in the previous month, availability for 24-hour close follow-up, and agreement to give a written informed consent (Table). The subjects were guided to continue their daily schedule but to avoid strenuous activity during the study period.

The study was conducted in 2 phases. The first phase was intended to characterize the WBC and the differential blood counts over 24 hours after a single-dose administration of inhaled CS. Subjects were randomly assigned to receive either 1 mg of budesonide inhalation in 2 mL of 0.9% saline using a face mask (study group A) or inhalation of 2 mL of 0.9% saline (group B, control). A control group was recruited, Phase 1: 24-hour effect of budesoni”>for comparison to the study group, to overcome diurnal variations in WBC. The inhalation of CS or saline was given in the morning between 8:00 and 10: 00 AM. Blood specimens were drawn from the antecubital vein at times 0 (just before inhalation), 6, 12, and 24 hours after inhala- tion and assayed for complete and differential blood count.

Phase 1 of the study demonstrated that the maximal effect of inhaled CS on WBC was after 6 hours (as is detailed in the “Results” section). Therefore, in the second phase of the study, blood specimens were drawn at 0 and 6 hours after inhalation. The additional volunteers were randomized into 3 groups: group C received 1 mg budesonide by inhalation using a face mask, group D received 200 ug budesonide using an active inhaler, and group E received fluticasone 250 ug using an active inhaler. Blood specimens were drawn from the antecubital vein and assayed for complete and differential blood counts, granulocyte colony-stimulating factor (G-CSF) level, and expression of neutrophils Mac-1 (CD11b) and L-selectin (CD62L).

Assays

Complete blood count including total leukocyte counts and differen- tial were determined on EDTA-anticoagulated blood specimens using an Advia 2120 Counter (Siemens, Erlangen, Germany).

Neutrophil adhesion molecule expression

L-selectin and Mac-1 expression on neutrophils was determined by immune fluorescence flow cytometry as previously described [13]. Briefly, direct immunofluorescence labeling of heparinized whole blood samples with CD62L and CD11b (fluorescein isothiocyanate; Beckman Coulter, Marseille, France) was performed following erythro- cyte lysis (FACS Lysing Solution; BD Biosciences San Jose, CA). Flow cy- tometry analyses were performed on a BD FACS Canto II cytometer (BD Biosciences). Ten thousand neutrophils, identified on the basis of light- scattering properties, were analyzed for adhesion molecule expression.

G-CSF assay

G-CSF concentration was determined in serum samples by enzyme- linked immunosorbent assay (Human G-CSF Instant ELISA; eBioscience, Vienna, Austria) according to manufacturer’s instructions.

Statistical analysis

The data of each individual at time zero were regarded as baseline and designated as 100%; results of blood specimens drawn later were expressed relative to the baseline. Continuous variables were presented as means +- SEM or median and interquartile range as appropriate. WBC, ANC, G-CSF level, and neutrophil Mac-1 and L-selectin expression were compared between the treatment groups using 1-way analysis of variance test or ?2 test, as appropriate. Analyses were performed by the SPSS 17 statistical software (IBM Corp, Chicago, IL). P value b .05 was a priori defined as significant.

Results

Sixty healthy volunteers (aged 21-48 years, 23 men) were recruited to the 2 phases of the study and randomized to the 5 study groups. All recruits completed the study protocol.

Phase 1: 24-hour effect of budesonide vs saline inhalation on WBC

Twenty subjects were included in this phase of the study: study group A (n = 10) received 1 mg of budesonide inhalation in 2 mL of 0.9% saline using a face mask, and the control group B (n = 10) received inhalation of 2 mL of 0.9% saline. The WBC and ANC following a single- dose inhalation of 1 mg budesonide are shown in Fig. 1. Six hours after budesonide inhalation, WBC increased by a mean of 23.4% (95% confi- dence interval [CI], 11.3-35.4), significantly more than in the saline con- trol group (4.1%; 95% CI, -5 to 13.2; Pb .01). The WBC increased in all 10 individuals who received inhaled budesonide. The mean increase in ANC after budesonide inhalation was greater than that in WBC (30.1%; 95% CI, 7.2-53.0) and significantly greater than that in the saline control group (6.1%; 95% CI, -2.5 to 14.7; Pb .01). No statistically significant differences from baseline were observed in the absolute lymphocyte

Table

Subject’s characteristics

Group

n

Age (y), mean +- SD

Gender, M:F ratio

Background disease

Smoking

A: Budesonide, face mask

10

32.3 +- 6.4

3:7

None

No

B: Control, saline inhalation

10

33.8 +- 7.7

3:7

None

No

C: Budesonide, face mask

20

29.3 +- 4.5

9:11

None

No

D: Budesonide, inhaler

10

30.0 +- 4.6

5:5

None

No

E: Fluticasone, inhaler

10

30.4 +- 3.1

3:7

None

No

Total

60

30.7 +- 5.7

23:37

0

0

P

.69

.41

Phase 2: effect of various inhaled “>Fig. 1. WBC and ANC following a single-dose inhalation of 1 mg budesonide.

or eosinophil counts (P= .14 and .17, respectively). The percentage of neutrophils of the total leukocyte count increased in the budesonide group from 54.6% to 58.1% (Pb .001), whereas the percentage of lymphocytes decreased from 34.3% to 31.9% (P= .014). No statistically significant differences were observed between control and study groups in WBC and ANC at 12 and 24 hours after inhalation.

Phase 2: effect of various inhaled steroids and inhalation techniques on WBC

Forty additional volunteers were included in this phase of the study in 3 randomized groups: group C (n = 20) received 1 mg budesonide by inhalation using a face mask, group D (n = 10) received 200 ug budesonide using an active inhaler, and group E (n = 10) received fluticasone 250 ug using an active inhaler. As the significant increases in WBC and ANC were noted 6 hours after inhalation, in the second phase of the study, blood specimens were taken at baseline (time 0) and at 6 hours after inhalation; the values at 6 hours were compared with those at baseline. Mean WBC and ANC increased for all inhaled-CS groups at 6 hours compared with baseline (Fig. 2), often with statistical significance, as detailed below. Administration of fluticasone by an active inhaler resulted in a mean 12.6% (95% CI, 1.5-23.7) increase in WBC (Pb .01) and a mean 22.7% (95% CI, 6.2-39.2) increase in ANC (Pb .01). After administration of 200 ug budesonide by an active inhaler, increases in WBC and ANC did not reach statistical significance (6.1%; 95% CI, -3.1 to 15.3; P= .41 and 13.4%; 95% CI, -0.3 to 22.6; P= .15,

respectively). No statistically significant differences were observed in changes from baseline of absolute lymphocyte or eosinophil counts in all study groups (P= .72 and .16, respectively).

The effect of inhaled CSs on the expression of adhesion neutrophil ligands and serum G-CSF levels

Neutrophil Mac-1 (CD11b) and L-selectin (CD62L) expression de- creased in all inhaled-CS groups at 6 hours compared with baseline (Fig. 3), often with statistical significance, as detailed below. Mac-1 ex- pression decreased by 51% (95% CI, 35.5-66.5; Pb .01) and 39.8% (95% CI, 2.7-76.9; P= .01) after inhalation of budesonide (face mask) and fluticasone (inhaler), respectively. L-selectin expression decreased after face mask inhalation of budesonide by 30.9% (95% CI, 11.9-49.7; P= .02). A nonstatistically significant reduction in L-selectin expression was observed in the fluticasone group (mean -17.4%; 95% CI, -10.1 to 44.9; P= .17). No statistically significant decreases in Mac-1 or L-selectin expression were noted after administration of budesonide by an inhaler (P= .55 and P= .47, respectively) or regarding the differences among the various inhaled-CS groups (P= .38 for both). Serum G-CSF levels did not change significantly from baseline in any of the groups (Fig. 3).

Limitations

Our study has several limitations; we assessed the effect of inhaled CSs on WBC and ANC using a number of point estimates (0, 6, 12, and 24 hours) rather than a continuoUS time effect, expressed by area under the curve. It is questionable whether asthmatic patients on steroids, who might be downregulated in their response to inhaled CSs, will respond similar to the healthy adults that were studied. Also, the sample sizes for the various groups were relatively small. However, this study was not designed to compare the various preparations but rather to highlight the contribution of inhaled CSs to the increased leukocyte and neutrophil counts, especially in the setting of the ED.

Discussion

The present study demonstrated that administration of conventional doses of currently used inhaled CSs significantly increased the WBC. This effect was attributed mainly to an increase in the ANC; thus, the percentage of neutrophils of the total leukocyte count increased signif- icantly, whereas the percentage of lymphocytes decreased significantly. These effects were seen in all inhaled-CS groups, although the amount of change and the level of statistical significance varied according to the specific CS given and the method of inhalation.

To the best of our knowledge, this is the largest randomized controlled study that examined the effect of inhaled CSs on the total and differential leukocyte count and that elucidated the mechanisms involved. Previous studies, performed mainly with systemic–oral or intravenous–administration of CSs, showed clear dose-dependent increases in WBC and ANC [8-11]. Thus, the influence of systemic CSs on the leukocyte count is well established.

Although inhaled CSs are widely and increasingly used in modern medicine, studies on their effects on WBC and ANC are limited by small sample sizes and lack of data on currently used inhaled CSs. More- over, findings are inconclusive because of equivocal results. Blaiss et al

[20] examined the effect of beclomethasone inhalation on leukocyte

count in volunteers, without a control group. They found that after inhalation of 0.4 mg beclomethasone, in 11 subjects, WBC and ANC increased significantly after 6 hours, with no significant changes in the lymphocyte or eosinophil counts. Cameron et al [24] examined the effect of inhaled beclomethasone and oral prednisolone on leuko- cyte count, without a controlled group. After 1-mg inhalation of beclomethasone, the percentage of neutrophils of the total leukocyte count increased by 8.5% from baseline in 8 healthy volunteers. The find- ings of those studies are in accordance with ours, although they did not investigate the currently used inhaled CSs, budesonide and fluticasone. In contrast, Brown et al [25] found no significant increase in ANC follow- ing inhalation of beclomethasone or budesonide as compared with con- trols; but that study comprised only 9 participants.

None of the 3 studies mentioned above investigated the mecha- nisms involved in the change of the leukocyte count after inhalation of CSs. Demargination of neutrophils from endothelial walls into the circu- lating pool has been shown to be the main route for increased neutro- phil count in the context of systemic CS administration [12,15]. Mac 1 (CD11b) and L-selectin (CD62L) have crucial roles in the initial attach- ment of circulating neutrophils to the vascular endothelium during the initiation of polymorphonuclear leukocyte recruitment to the sys- temic inflammatory site through cellular signaling [13,15].

The current study, to our knowledge, is the only one that examined

the mechanisms involved in inhaled CS-induced increased leukocyte count. We found that the expression of both adhesion neutrophil li- gands Mac-1 and L-selectin decreased in all inhaled-CS groups com- pared with baseline. This concurs with previous studies performed with systemic CS administration and also with experimental studies that showed increased neutrophil and leukocyte counts related to de- tachment of cells from vascular walls [12-15]. Bone marrow production was reported to have a minor contribution to increased neutrophil and

Fig. 2. Mean WBC (A) and ANC (B) at 6 hours compared with baseline.

leukocyte counts after systemic administration of CS [13]. Therefore, we examined serum G-CSF levels before and after inhalation of CSs yet found no significant changes. Other less contributing and less likely mechanisms were not examined.

The current findings of inhaled CS-induced increases in WBC and ANC and of decreased expression of neutrophil adhesion molecules, which are similar to observations following systemic CS administration, reinforce the probability that such effects are related to systemic ab- sorption of inhaled CSs. Indeed, minimal systemic absorption of inhaled CSs has been documented, with effects on the hypothalamic-pituitary-

Fig. 3. .G-CSF, CD11b, and CD62L at 6 hours compared with baseline.

adrenal axis and on body metabolism [19,20,23]. The degree of absorption depends on both the specific CS administered and the method of inhalation.

The doses administered in this study were selected based on the range of doses commonly administered for acute wheezing in toddlers and preschool children, or maintenance therapy doses for mild forms of childhood asthma [26,27]. However, no attempt was made to com- pare the response of the various inhaled CS preparations.

The maximal increase in neutrophil counts was observed in the fluticasone (active inhaler) group, of which the selected dose was in the mid-high range of the commonly used doses. In contrast, in the budesonide (active inhaler) group, for which the dose given was in the mid-low range of the commonly administered dose, no statistical in- crease was demonstrated. This observation suggests a dose-dependent relationship of inhaled CS and leukocyte count.

It should be mentioned that although we documented a significant increase in WBC, no overt leukocytosis was demonstrated. This fact does not reduce the importance of our findings, as we believe that an understanding of the effect of recent inhaled CS administration on the total and differential leukocyte count has important clinical relevance and should be considered in the interpretation of white blood counts. Moreover, it is plausible that serial inhalations, higher doses, and con- comitant use of other medications, as short-acting ?-agonists, may ag- gravate a greater increase in ANC and WBC.

In summary, CS inhalations that are currently widely used result in increases in WBC and ANC. As the neutrophil and leukocyte counts are important in decision making, for example, in the scenario of a patient with wheezing attending the ED after receiving inhaled CS, clinicians should be aware of this effect and interpret the complete blood count accordingly. Future studies are planned to evaluate the additive effects of additional treatments given in the ED and of serial inhalations, with particular attention to specific populations such as children, who may have a potentially more pronounced response due to higher intestinal absorption of inhaled CSs. Studies of asthmatic patients, especially those who are under Steroid therapy, are also needed.

References

  1. Riley LK, Rupert J. Evaluation of patients with leukocytosis. Am Fam Physician 2015; 92(11):1004-11.
  2. Aydin Sunbul E, Sunbul M, Yanartas O, et al. Increased neutrophil/lymphocyte ratio in patients with depression is correlated with the severity of depression and Cardiovascular risk factors. Psychiatry Investig 2016;13(1):121-6.
  3. Dogru M, Yesiltepe Multu RG. The evaluation of neutrophil-lymphocyte ratio in children with asthma. Allergol Immunopathol 2016 [Epub ahead of print].
  4. Abramson N, Melton B. Leukocytosis: basics of clinical assessment. Am Fam Physician

    2000;62(9):2053-66.

    Ishimine P. fever without source in children 0 to 36 months of age. Pediatr Clin North Am 2006;53(2):167-94.

  5. Bonsu BK, Harper MB. Utility of the peripheral blood white blood cell count for identifying sick young infants who need lumbar puncture. Ann Emerg Med 2003; 41(2):206-14.
  6. Bonsu BK, Chb M, Harper MB. Identifying febrile young infants with bacteremia: is the peripheral white blood cell count an accurate screen? Ann Emerg Med 2003; 42(2):16-25.
  7. Mintzer DM, Billet SN, Chmielewski L. Drug-induced hematologic syndromes. Adv Hematol 2009;2009:1-11, 495863.
  8. Dale DC, Fauci AS, Guerry IV D, Wolff SM. Comparison of agents producing a neutro- philic leukocytosis in man. J Clin Invest 1975;56(4):808-13.
  9. Shoenfeld Y, Gurewich Y, Gallant LA, Pinkhas J. Prednisone-induced leukocytosis. In- fluence of dosage, method and duration of administration on the degree of leukocy- tosis. Am J Med 1981;71(5):773-8.
  10. Youssef P, Roberts-Thomson P, Ahern M, Smith M. Pulse methyl prednisolonein rheumatoid arthritis: effects on peripheral blood and synovial fluid neutrophil sur- face phenotype. J Rheumatol 1995;22(11):2065-71.
  11. Nakagawa M, Terashima T, D’yachkova Y, Bondy GP, Hogg JC, van Eeden SF. Glucocorticoid-induced granulocytosis: contribution of marrow release and demargination of intravascular granulocytes. Circulation 1998;98(21):2307-13.
  12. Ad C, Boylan MT, Droogan AG, McMillan SA, Hawkins SA. Methylprednisolone- induced neutrophil leukocytosis–down modulation of neutrophil L-selectin and Mac-1 expression and induction of granulocyte-colony stimulating factor. Int J Clin Res 1998;28(2):110-5.
  13. Bishop CR, Athens JW, Boggs DR, Warner HR, Cartwright GE, Wintrobe MM. Leukokinetic studies: a non-steady-state kinetic evaluation of the mechanism of cortisone-induced granulocytosis. J Clin Invest 1968;47(2):249-60.
  14. Ivetic A. Signals regulating L-selectin-dependent leucocyte adhesion and transmi- gration. Int J Biochem Cell Biol 2013;45(3):550-5.
  15. Liles WC, Dale DC, Klebanoff SJ. Corticosteroids inhibit apoptosis of human neutro- phils. Blood 1995;86(8):3181-8.
  16. Daley-Yates PT. Inhaled corticosteroids: potency, dose equivalence and therapeutic index. Br J Clin Pharmacol 2015;80(3):372-80.
  17. Passalacqua G, Albano M, Canonica GW, et al. Inhaled and nasal corticosteroids: safe- ty aspects. Allergy 2000;55(1):16-33.
  18. Fitzgerald D, Van Asperen P, Mellis C, Honner M, Smith L, Ambler G. Fluticasone pro- pionate 750 micrograms/day versus beclomethasone dipropionate 1500 micro- grams/day: comparison of efficacy and adrenal function in paediatric asthma. Thorax 1998;53(8):656-61.
  19. Blaiss MS, Herrod HG, Crawford LV, Lieberman PL. Beclomethasone dipropionate aerosol: hematologic and immunologic effects. Ann Allergy 1982;48(4):210-4.
  20. Ducharme FM, Lemire C, Noya FJ, et al. Preemptive use of high-dose fluticasone for virus-induced wheezing in young children. N Engl J Med 2009;360(4):339-53.
  21. Kaliner MA. Pharmacologic characteristics and Adrenal suppression with newer in- haled corticosteroids: a comparison of ciclesonide and fluticasone propionate. Clin Ther 2006;28(3):319-31.
  22. Monson JP. Systemic effects of inhaled corticosteroids. Thorax 1993;48(10):955-6.
  23. Cameron RG, Black PN, Braan C, Browett PJ. A comparison of the effects of oral pred- nisone and inhaled beclomethasone dipropionate on circulating leukocytes. Aust N Z J Med 1996;26(6):800-5.
  24. Brown PH, Matusiewicz SP, Shearing C, Tibi L, Greening AP, Crompton GK. Systemic effects of high dose inhaled steroids: comparison of beclomethasone dipropionate and budesonide in healthy subjects. Thorax 1993;48(10):967-73.
  25. Brand PL, Caudri D, Eber E, et al. Classification and pharmacological treatment of pre- school wheezing: changes since 2008. Eur Respir J 2014;43(4):1172-7.
  26. Volovitz B. Inhaled budesonide in the management of acute worsenings and exacerbations of asthma: a review of the evidence. Respir Med 2007;101(4): 685-95.