Biologically active neutrophil chemokine pattern in tonsillitis

Abstract

To gain an insight into the mechanisms of chronic and acute inflammation, the production of neutrophil chemokines in different types of tonsillitis - hyperplastic tonsillitis (HT), recurrent tonsillitis (RT) and peritonsillar abscesses (PA) - was investigated. The chemokines interleukin-8 (IL-8), growth-related oncogene-alpha (GRO-alpha), epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2) were detected and shown to have different biological activities. With respect to the biological properties of CXC chemokines, the biological activity of the chemokines was identified using a three-step high-performance liquid chromatography (HPLC) technique, a bioassay involving measurement of neutrophil chemotaxis in a single Boyden chamber in tissue of HT, RT and PA. Using reverse transcription-polymerase chain reaction (RT-PCR), the chemokine concentrations were determined in the different tonsillitis entities. The chemokine pattern was dominated in PA by IL-8 and GRO-alpha and in RT by GRO-alpha. Hyperplastic tonsils of patients without a history of infection generated about five times lower IL-8 than PA. A protein concentration of GCP-2 was induced in PA and RT, whereas ENA-78 remained the same in all entities. In conclusion, it would appear that IL-8 was up-regulated in acute inflammation, whereas GRO-alpha dominated in chronic inflammation. ENA-78 seems not to play a pivotal role in inflammatory processes in tonsils. GCP-2 may serve as a substitute chemokine in certain inflammatory conditions as its quantity of mRNA and protein was higher in RT and PA than in HT.

Fig. 1

Chemotactic activity of proteins after reversed-phase chromatography (C8 nucleosil RP-8) in hyperplastic tonsils. (a) Chromatogram (RP-8 column). Supernatant collected from tissue homogenization was concentrated and applied to a heparin–sepharose cartridge. The eluate was collected as one fraction and subjected to size exclusion chromatography (RP-8 column). The figure shows the chromatogram and the collected protein fractions labelled with numbers 1–14. (b) Chemotactic index and concentrations of chemokines. The manually collected protein fractions 1–14 were then analysed for chemotactic activity. In each test series, both positive and negative controls were included at appropriate concentrations. FNLP (formyl-Nle-Leu-Phe) served as a positive control for chemotactic responsiveness of PMN, whereas PBS was used as a negative control. The data shown here are those corresponding to 30 µl fraction aliquots transferred to the Boyden chamber. All fraction volumes tested had been freeze-dried prior to testing. In addition, an ELISA was performed to identify the different chemokines. The corresponding fractions were pooled as fractions 1, 2, 3 and 4 and applied to a Mono-S column (see Fig. 2).

Fig. 2

Chromatogram of microcation exchange HPLC and chemotactic index of the protein fractions from HT. Bioactive fractions from the RP-8 column (1–5 = fraction number 1; 6–7 = fraction number 2; 9–10 = fraction number 3; 11–14 = fraction number 4) were applied to the Mono-S column. Fractions from Mono-S runs were collected automatically and again tested for chemotactic activity (10 µl and 60 µl aliquot protein fractions were collected for the Boyden chamber test). Two chromatograms are shown as examples: run number 2 in (a) and run number 3 in (b), with chemotactic indices and ELISA measurements identifying the chemokines.

Fig. 3

Chemotactic activity of proteins after reversed-phase chromatography (C8 nucleosil RP-8) in recurrent tonsillitis. (a) Chromatogram (RP-8 column). Supernatant collected from tissue homogenization was concentrated and applied to a heparin–sepharose cartridge. The eluate was collected as one fraction and subjected to size exclusion chromatography. The figure shows the chromatogram and the collected protein fractions labelled with numbers 1–18. (b) Chemotactic index and concentrations of chemokines. The manually collected protein fractions 1–18 were then analysed for chemotactic activity. In each test series, both positive and negative controls were included at appropriate concentrations. FNLP (formyl-Nle-Leu-Phe) served as a positive control for chemotactic responsiveness of PMN, whereas PBS was used as a negative control. The data shown here are those corresponding to 30 µl fraction aliquots transferred to the Boyden chamber. All fraction volumes tested had been freeze-dried prior to testing. In addition, an ELISA was performed to identify the different chemokines. Corresponding fractions were pooled as fractions 1, 2, 3 and 4 and subjected to Mono-S column (see Fig. 4).

Fig. 4

Chromatogram of microcation exchange HPLC and chemotactic index of the protein fractions from RT. Bioactive fractions from the RP-8 column were pooled into four fractions and applied to the Mono-S column. Fractions from Mono-S runs were collected automatically and again tested for chemotactic activity (10 µl and 60 µl aliquot protein fraction were collected for the Boyden chamber test). Two chromatograms are shown as examples: run number 2 in (a) and run number 3 in (b), with chemotactic indices and ELISA measurements identifying the chemokines.

Fig. 5

Chemotactic activity of proteins after reversed-phase chromatography (C8 nucleosil RP-8) in peritonsillar abscesses. (a) Chromatogram (RP-8 column). Supernatant collected from tissue homogenization was concentrated and applied to a heparin–sepharose cartridge. The eluate was collected as one fraction and subjected to size exclusion chromatography. The manually collected protein fractions 1–14 were then analysed for chemotactic activity. The figure shows the chromatogram and the collected protein fractions labelled with numbers 1–18. (b) Chemotactic index and concentrations of chemokines. In each test series, both positive and negative controls were included at appropriate concentrations. FNLP (formyl-Nle-Leu-Phe) served as a positive control for chemotactic responsiveness of PMN, whereas PBS was used as a negative control. The data shown here are those corresponding to the 30 µl fraction aliquots transferred to the Boyden chamber. All fraction volumes tested were freeze-dried prior to testing. In addition, an ELISA was performed to identify the different chemokines. Corresponding fractions were pooled as fractions 1, 2, 3 and 4 and subjected to a Mono-S column (data are not shown).

Fig. 6

Chemokine mRNA expression in HT, RT and PA. Chemokine mRNA levels are expressed as a quotient of chemokine CT value/GAPDH CT value (mean ± s.e.m.). Significant results are marked with an asterisk (P < 0·05).