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. 1999 Aug;117(2):237–243. doi: 10.1046/j.1365-2249.1999.00982.x

Increased levels in vivo of mRNAs for IL-8 and macrophage inflammatory protein-1α (MIP-1α), but not of RANTES mRNA in peripheral blood mononuclear cells of patients with atopic dermatitis (AD)

Y Hatano 1, K Katagiri 1, S Takayasu 1
PMCID: PMC1905337  PMID: 10444253

Abstract

Chemokines play an important role in the selective movement of leucocytes into inflammatory areas and they also activate various cells in inflamed tissues. However, it is unclear which cells are the main sources of chemokines in actual inflammatory diseases, even though both mononuclear cells and non-inflammatory resident cells are able to produce chemokines in vitro and the former cells are also the main target of chemokines. To clarify the roles of chemokines that are produced by mononuclear cells in AD, we measured levels in vivo of mRNA for IL-8 and MIP-1α, as well as the level of regulated upon activation normal T cell expressed and secreted (RANTES) mRNA in freshly isolated peripheral blood mononuclear cells from patients with AD. We compared the results with those from psoriatic patients, and patients without AD who were suffering from other cutaneous diseases and eosinophilia. Levels of mRNAs were determined by semiquantitative reverse transcriptase-polymerase chain reactions. Levels of IL-8 and MIP-1α mRNA were elevated not only in atopic patients but also in non-atopic patients with inflammatory skin disease associated with eosinophilia, compared with levels in psoriatic patients and healthy controls. Levels of RANTES mRNA were similar in atopic patients but they were lower in the other two groups of patients when compared with levels in healthy controls. In atopic patients, the levels of both IL-8 and MIP-1α mRNAs but not of RANTES mRNA decreased with improvements in symptom scores after therapy. These findings suggest that mononuclear cells are not only the target of chemokines but might also play an important role in the pathogenesis of AD by producing IL-8 and MIP-1α.

Keywords: IL-8, macrophage inflammatory protein-1α, RANTES, mononuclear cells, atopic dermatitis

INTRODUCTION

Chemokines play an important role in the selective movement of leucocytes into areas of inflammation [1]. IL-8, which is a member of the so-called ‘C-X-C’ subfamily of chemokines, is a chemotactic attractant for neutrophils. Moreover, MIP-1α and the chemokine known as regulated upon activation normal T cell expressed and secreted (RANTES), which are ‘C-C’ chemokines, are chemotactic attractants for lymphocytes, monocytes, and eosinophils. These chemokines can activate or regulate the activities of the various kinds of cells that make up inflamed tissues [1–4]. These chemokines are produced not only by non-inflammatory resident cells, such as epithelial cells, fibroblasts, keratinocytes, and endothelial cells [5–7], but also by monocytes and lymphocytes, which are also major targets of the chemotactic activity of these chemokines [8]. Some previous studies have suggested the participation of IL-8 and RANTES in the pathogenesis of inflammatory skin diseases, such as AD [9–12], psoriasis vulgaris [7,13–15], and bullous pemphigoid [16,17]. However, the sources of the chemokines in diseases of the skin have not yet been fully clarified. To our knowledge, there have been no reports on the relationship between MIP-1α and cutaneous diseases.

In this study, we measured levels of mRNAs for IL-8, RANTES and MIP-1α in peripheral blood mononuclear cells (PBMC) by semiquantitative reverse transcriptase-polymerase chain reactions (RT-PCR) [18,19] in an attempt to determine whether the production of chemokines by mononuclear cells plays a role in the pathogenesis of AD. We also measured levels of the mRNAs for these chemokines in patients with other cutaneous diseases with eosinophilia. Patients with psoriasis vulgaris were selected as controls because Th1 cytokines are expressed in the skin lesions of these patients, in contrast to the Th2 cytokines expressed in AD [20,21]. We also examined whether topical corticosteroids might affect the symptoms of AD by modulating the production of these chemokines in mononuclear cells in vivo.

PATIENTS AND METHODS

Study populations

Details of the patients enrolled in this study are shown in Table 1. Informed consent was obtained from all patients and from healthy controls. All patients with AD fulfilled the diagnostic criteria of Hanifin & Rajka [22]. Thirteen patients with AD had personal histories of respiratory allergy which was mild or inactive. The 11 patients with non-atopic dermatosis with eosinophilia included three patients with bullous pemphigoid, three with drug eruption, two with chronic dermatitis, one with erythroderma of unknown origin, one with adult T cell leukaemia, and one with hypereosinophilic syndrome. All psoriatic patients were diagnosed on the basis of typical histological findings, which included psoriatic acanthosis and Munro's microabscesses, with eruptions affecting 10–40% of the body surface. None of these non-atopic patients and healthy individuals had any history of atopic disease. Among the patients with AD, five were classified as having mild disease, 10 as having moderate disease, and 12 as having severe disease, according to the scoring system of Rajka & Langeland [23]. In addition, we scored the symptoms of AD as follows. Intensities of eruption were evaluated as 0, none or dry skin only; 1, erythema with or without scaling; 2, 1 plus excoriation or papules; and 3, 1 or 2 plus lichenification or impetigo. Total symptom scores were calculated by multiplying intensities of eruption by the percentage of the body surface involved, so that scores ranged theoretically from 0 to 300. The mean symptom score was 140 (range 18–243). Heparinized blood samples were collected from all patients during exacerbation of eruptions. Patients with AD or with psoriasis vulgaris were only receiving topical corticosteroids infrequently when blood samples were obtained. In 12 atopic patients, whose mean symptom score was 150 (range 81–198) before treatment, blood samples were obtained again when symptoms had improved (mean symptom score 45; range 18–81) after treatment with topical corticosteroids and systemic azelastin hydrochloride (mean duration of treatment 60 days; range 5–211 days).

Table 1.

Mean values with ranges of the ages, serum IgE levels and eosinophil counts of patients and healthy controls (HC)

graphic file with name cei0117-0237-t1.jpg

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M, Male; F, female. Numbers in parentheses represent ranges.

Isolation of RNA from PBMC

Mononuclear cells were isolated from heparinized venous blood by centrifugation over Ficoll–Paque (Pharmacia, Uppsala, Sweden). They were immediately homogenized in Isogen (Nippon Gene, Toyama, Japan), which contained phenol and acid guanidinium thiocyanate. Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method [24].

Semiquantitative RT-PCR

Spontaneous expression of mRNAs for IL-8, MIP-1α, RANTES and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; for normalization of results) in freshly isolated PBMC was measured by semiquantitative RT-PCR as described previously [18]. Total RNA (50 ng) was reverse-transcribed with random hexamers and MMLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD). The resultant cDNA was amplified by PCR with or without 1 mCi 32P-dCTP. Amplification was allowed to proceed for 16–38 cycles (1 min at 94°C, 1 min at 58°C, 1 min at 72°C) for each pair of primers (see also below). The oligonucleotide primers used for PCR were based on published sequences of mRNAs. The primers for amplification of cDNA for human G3PDH were 5′-CCCATCACCATCTTCCAG-3′ as the upstream primer and 5′-CCTGCTTCACCACCACCTTCT-3′ as the downstream primer; for IL-8, they were 5′-ACAAGCTTCTAGGAC AAGAGCC-3′ as the upstream primer and 5′-ACTTCTCCACAACCCTCTGC-3′ as the downstream primer; for MIP-1α, they were 5′-TCACCTGCTCAGAATCATGC-3′ as the upstream primer and 5′-TCCATAGAAGAGGTAGCTGTGG as the downstream primer; and for RANTES, they were 5′-ATGAAGGTCTCCGCGGCACGCCT-3′ as the upstream primer and 5′-CTAGCTCATCTCCAAAGAGTTG-3′ as the downstream primer.

We identified the products of PCR as follows. Each reaction mixture was fractionated by electrophoresis on an agarose gel, and bands of amplified cDNA were extracted and cDNA were digested with appropriate restriction endonucleases. Mobilities were compared with those of 100-bp molecular size markers (Gibco-BRL) and the sizes of the products of PCR and their fragments were compared with the expected values. Aliquots (1 μl) of each sample prepared with 32P-dCTP were analysed by electrophoresis on 5% acrylamide/Tris-borate EDTA gels and subsequent autoradiography. DNA was quantified by phosphor image analysis (BAS2000; Fuji Medical Systems, Stanford, CT). Linear ranges for amplification with each pair of primers were determined by reference to the number of cycles of amplification and the amount of cDNA. Several samples were subjected to PCR to exclude the possibility that decreased amounts of a specific cDNA might have led to interference with the linearity of the rate of amplification by PCR. The appropriate PCR cycles within the linear amplification ranges were performed as follows: 20 cycles for G3PDH, 22 cycles for RANTES, 25 cycles for IL-8, and 28 cycles for MIP-1α.

Statistical analysis

The intensities of amplified signals were normalized by reference to the signal derived from G3PDH mRNA, which was used as the internal standard. Results were compared and evaluated using a variance test (Kruskal–Wallis test), Mann–Whitney U-test or Wilcoxon's signed ranks test. Correlation coefficients and statistical significance were determined using Spearman's rank correlation coefficient. P < 0.05 was considered statistically significant.

RESULTS

The mobilities of the products of PCR and the lengths of their restriction fragments corresponded closely to the values predicted for each pair of primers (116 bp and 222 bp for IL-8; 99 bp and 305 bp for MIP-1α; and 233 bp and 42 bp for RANTES) (Fig. 1). Samples of RNA that had not been reverse-transcribed did not generate amplified cDNAs with any of the primer pairs. As shown in Fig. 2, the intensities of signals of the products of RT-PCR were linearly related to the amounts of RNA that were included in the initial RT reaction.

Fig. 1.

Fig. 1

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Products of the polymerase chain reaction (PCR) and their restriction fragments after electrophoresis on a 2% agarose gel. Lanes 1, 4, and 7, 100-bp ladder; lanes 2, 5, and 8, cDNA for IL-8, MIP-1α, and regulated upon activation normal T cell expressed and secreted (RANTES), respectively, before digestion; lane 3, cDNA for IL-8 after digestion with PstI; lane 6, cDNA for MIP-1α after digestion with HindIII; lane 9, cDNA for RANTES after digestion with XcmI.

Fig. 2.

Fig. 2

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Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of mRNA. Each panel shows the radioactivity of products amplified by PCR plotted against increasing amounts of sample for each primer pair. 32P-dCTP was incorporated into the products of PCR, which were subjected to electrophoresis in polyacrylamide gels. The results were analysed by phosphor image analysis after autoradiography. Photostimulated luminescence (PSL) indicates the original value obtained by phosphor image analysis.

Expression of mRNAs for IL-8 and MIP-1α in PBMC from patients with AD, psoriasis vulgaris, non-atopic dermatosis with eosinophilia, and healthy controls

Expression of mRNAs for both IL-8 and MIP-1α was detected in all samples (Fig. 3). The levels of expression of the mRNAs for these two chemokines changed in parallel in each patient. Moreover, the levels of mRNAs for these two chemokines in atopic patients and in non-atopic patients with eosinophilia were significantly higher than those in healthy controls (Kruskal–Wallis test: IL-8, P = 0.0008; MIP-1α, P = 0.0053; Mann–Whitney U-test: atopic, P = 0.008 and P = 0.02; non-atopic, P = 0.002 and P = 0.046, for IL-8 and MIP-1α, respectively) as shown in Fig. 4a,b. There was no significant difference in the respective levels of IL-8 and MIP-1α mRNAs between atopic patients and non-atopic patients with eosinophilia (Fig. 4a,b). Among the atopic patients, those with eosinophilia tended to have higher levels of IL-8 and MIP-1α mRNAs than those without eosinophilia (with eosinophilia, mean relative levels 6.0 and 4.6; without eosinophilia, mean relative levels 1.4 and 1.1, respectively), but the differences were not statistically significant. Levels of IL-8 and MIP-1α mRNAs in atopic patients decreased significantly with decreases in symptom scores after therapy (Wilcoxon signed ranks test, P = 0.005 and P = 0.0096, respectively; Fig. 5a,b,d). In psoriatic patients, levels of IL-8 and MIP-1α mRNAs were lower than those in atopic patients (Mann–Whitney U-test, P = 0.008 and P = 0.005, respectively), in non-atopic patients with eosinophilia (Mann–Whitney U-test, P = 0.04 and P = 0.02, respectively) and in healthy controls (Mann–Whitney U-test, NS and P = 0.0496, respectively) (Fig. 4a,b). However, in two patients with severe psoriasis, levels of IL-8 and MIP-1α mRNAs were elevated. By contrast, the clinical severity scores and symptom scores of atopic patients was not correlated with the levels of mRNA for these chemokines (data not shown).

Fig. 3.

Fig. 3

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Autoradiogram showing results of amplification by reverse transcription-polymerase chain reaction (RT-PCR) of mRNAs for IL-8, MIP-1α, and regulated upon activation normal T cell expressed and secreted (RANTES) in peripheral blood mononuclear cells (PBMC) from five representative atopic patients before (lanes 1–5) and after therapy (lanes 6–10), from non-atopic patients with eosinophilia (lanes 11–15), from psoriatic patients (lanes 16–20), and from healthy controls (HC, lanes 21–25). The data shown represent rather extreme cases, so that differences can be easily recognized.

Fig. 4.

Fig. 4

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Comparison of levels of expression of mRNAs for IL-8 (a), MIP-1α (b), and regulated upon activation normal T cell expressed and secreted (RANTES) (c) in peripheral blood mononuclear cells (PBMC) from atopic patients, non-atopic patients with eosinophilia, psoriatic patients, and healthy controls (HC). The intensity of the signal corresponding to the level of mRNA for each chemokine was measured by phosphor image analysis and normalized by reference to that for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Medians are indicated by bold horizontal lines. Paired comparisons were made by Mann–Whitney U-test. NS, Not significant.

Fig. 5.

Fig. 5

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Normalized levels of mRNAs for IL-8 (a), MIP-1α (b), and regulated upon activation normal T cell expressed and secreted (RANTES) (c) in peripheral blood mononuclear cells (PBMC) from atopic patients before and after therapy. Changes in symptom scores are shown in (d). Results were compared and evaluated using Wilcoxon's signed ranks test. NS, Not significant.

Expression of RANTES mRNA in PBMC from patients with AD, psoriasis vulgaris, non-atopic dermatosis with eosinophilia, and healthy controls

The expression of RANTES mRNA was detectable in all samples by RT-PCR (Fig. 3). The levels did not differ significantly between healthy controls and atopic patients (Fig. 4c). The levels of RANTES mRNA in non-atopic patients with eosinophilia and in psoriatic patients were significantly lower than those in healthy controls (Kruskal–Wallis test: P = 0.01; Mann–Whitney U-test, P = 0.02 and P = 0.003, respectively; Fig. 4c), while the levels in these two groups of patients were not significantly different from those in atopic patients (Fig. 4c). There was also no significant difference in levels of RANTES mRNA between atopic patients with and without eosinophilia (data not shown). By contrast to levels of IL-8 and MIP-1α mRNAs, the change in levels of RANTES mRNA in atopic patients after therapy showed no clear tendencies (Fig. 5c). The levels of RANTES mRNA were also not correlated with disease severity and clinical scores in atopic patients (data not shown).

DISCUSSION

Our present study revealed that, in patients with AD and also in those with non-atopic cutaneous disease with eosinophilia, levels of IL-8 and MIP-1α mRNAs in PBMC were higher than those in psoriatic patients and healthy controls. In psoriatic patients, the levels of IL-8 and MIP-1α mRNAs in PBMC were similar to or lower than those in healthy controls. The results suggest that PBMC-derived IL-8 and MIP-1α might play a role in the pathogenesis of AD, even though elevated levels of these chemokines in PBMC were not specific to AD. By contrast, the level of MIP-1α mRNA was lower and that of IL-8 mRNA was normal in patients with psoriasis vulgaris, a Th1 disease. Thus, increased production of IL-8 and MIP-1α by PBMC might be characteristic of Th2 disease, if we assume that eosinophilia is associated with Th2 rather than with Th1 diseases. In studies in vitro, both Th1 and Th2 cell lines produced these chemokines upon appropriate stimulation [25,26]. Different sources and experimental conditions are probably responsible for differences in results. Alternatively, it is also possible that, independent of the Th1/Th2 balance, increases in levels of IL-8 and MIP-1α mRNAs might be associated with eosinophilia only, as was the case in atopic patients in the present study. In fact, eosinophilia does not necessarily reflect a Th2 dominant state: elevated levels of IL-5, which is a Th2 cytokine and the main factor associated with the differentiation and regulation of eosinophils, are associated with eosinophilia but not with increases in levels of IL-4, one of the other Th2 cytokines that is produced by antigen-specific T cells [27]. The present results do not exclude the possible participation of PBMC-derived IL-8 and MIP-1α in the pathogenesis of psoriasis vulgaris since, in two patients with severe psoriasis, the levels of the mRNAs for these chemokines were very high. Moreover, increased rates of spontaneous production of IL-8 in vitro by peripheral blood monocytes from psoriatic patients have been reported [13,14].

Although increased levels of RANTES protein and elevated expression of RANTES mRNA in inflamed tissues have been reported in patients with psoriasis vulgaris [7] and AD [11,12], we found that levels of RANTES mRNA in PBMC from patients with AD, non-atopic cutaneous disease with eosinophilia, and psoriasis vulgaris were similar to or lower than levels in PBMC from healthy controls. Similarly, Kodama et al. demonstrated elevated levels of RANTES mRNA in bronchoalveolar lavage cells but not in PBMC from patients with interstitial lung disease [28]. Expression of RANTES mRNA was reported to be markedly reduced in functional cytotoxic T cells in vitro, whereas it was elevated in freshly isolated peripheral lymphocytes after activation with an antigen [29]. PBMC include monocytes and lymphocytes, such as B cells, helper T cells and suppressor T cells. This mixture of various kinds of cell might somehow have been responsible for the reduction in levels of RANTES mRNA in PBMC from the patients in the present study. Furthermore, other cells, such as platelets [30,31], fibroblasts [6], endothelial cells [32], and keratinocytes [7], might also express RANTES protein in patients with cutaneous disease. Schroder et al. detected RANTES protein in supernatants of cultures of stimulated dermal fibroblasts, but not in those of T lymphocytes derived from atopic skin [12]. Further studies of the production of RANTES by mononuclear cells in inflamed areas are required to determine whether mononuclear cells are major sources of RANTES protein. The differences between levels of RANTES mRNA, and levels of IL-8 and MIP-1α mRNAs in PBMC observed in the present study might have been due to the different origins of the mRNAs. For example, IL-8 and MIP-1α might mainly be produced by monocytes, while RANTES protein might be produced by lymphocytes rather than by monocytes [33,34].

In the present study, the levels of mRNAs for IL-8 and MIP-1α in PBMC from atopic patients decreased as symptom scores improved upon administration of topical corticosteroids and systemic anti-histamines. Since corticosteroids can suppress the production of IL-8 and MIP-1α in mononuclear cells in vitro [35–38], it is possible that topically applied corticosteroid might directly affect the expression of mRNAs for these chemokines in PBMC. Alternatively, topical corticosteroids might indirectly suppress the expression of these mRNAs by PBMC through the action of mediators, such as keratinocyte-derived inflammatory cytokines. In addition, it is also possible that different populations of mononuclear phagocytes and subpopulations of lymphocytes or PBMC of different stages of maturity before and after corticosteroid therapy might have affected the expression of the mRNAs. We observed insignificant changes in the expression of RANTES mRNA after administration of corticosteroids. These drugs might suppress the abnormal expression of mRNAs for chemokine mRNAs but might not suppress the constitutive expression of these mRNAs. Corticosteroids might exert their effects on atopic patients, at least to some extent, by suppression of the expression of mRNAs for these chemokines. Finally, levels of neither IL-8 mRNA nor MIP-1α mRNA expression were correlated with disease severity or symptom scores in patients with AD. Our results suggest that additional factors are involved in the exacerbation of AD.

In conclusion, the present study suggests that IL-8 and MIP-1α, produced by mononuclear cells, might play a role in the pathogenesis of AD and of other various diseases of the skin that are accompanied by eosinophilia.

Acknowledgments

The authors are grateful to Professor N. Eshima and Associate Professor H. Aono of Oita Medical University for their valuable advice related to the statistical analysis of the data.

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