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Keywords:

  • LPS;
  • TNF-α;
  • IL-10;
  • Eosinophil

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Macrophage inflammatory protein-3α (MIP-3α) / CCL20 and MIP-3β / CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8 / CXCL8, GROα / CXCL1, IP-10 / CXCL10, and MIG / CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3α / CCL20 and MIP-3β / CCL19 when cultured with either LPS or TNF-α. We also report that MIP-3α / CCL20 and MIP-3β / CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3α / CCL20 and anti-MIP-3β / / CCL19 neutralizing antibodies, indicating that MIP-3α / CCL20 and MIP-3β / CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability ofneutrophils to produce MIP-3α / CCL20 and MIP-3β / CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to theregulation of the immune response.


1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Neutrophils are known to play an important role in inflammatory responses by virtue of their ability to perform a series of effector functions that collectively represent an essential homeostatic mechanism of innate immunity against injury and infection. In addition to their defensive functions, it is well established that neutrophils can synthesize and secrete several cytokines 1, including a number of chemokines, such as, for instance, IL-8 / CXCL8, growth-related gene product-α (GROα) / CXCL1, macrophage inflammatory protein-1α (MIP-1α) / CCL3,MIP-1β / CCL4, IFN-γ-inducible protein of 10 kDa (IP-10) / CXCL10, monokine induced by IFN-γ (MIG) / CXCL9, and IFN-inducible T cell alpha chemoattractant (I-TAC) /CXCL11 1 – 3. The findings that PMN are capable of producing several chemokines, and major proinflammatory cytokines as well, indicate that these cells may be important in directing cell trafficking during the initial phases of pathophysiological processes.

Recently, a number of chemokines have been discovered through bioinformatic technologies, including MIP-3α / CCL20 and MIP-3β / CCL19 4. MIP-3α / CCL20, also known as LARC or Exodus, has been shown to be a specific, high-affinity ligand for the CCR6 receptor 5, and to attract immature dendritic cells (DC) derived from CD34+ hemopoietic progenitors 5, 6, memory subset of T cells 7, and B cells 8. More recently, MIP-3α / CCL20 has been also found tobe chemotactic for CD11b+ myeloid DC in the Peyer's patch, and to be involved in the regulation of humoral immunity and lymphocyte homeostasis in the intestinal mucosa 9,10. MIP-3β / CCL19 is identical to Ck-beta-11 and the EBI-1-Ligand chemokine (ELC) 5, and binds exclusively to CCR7 on mature DC 5, 6, naive and TCR-activated effector / memory T cells 11, 12, 13, B cells 8, 11, 12, and NK cells 14, inducing the chemotaxis of all these cells. MIP-3α / CCL20 is constitutively expressed in appendix, tonsil, liver, placenta, mucosal and inflamed tissue and is inducible in activated hematopoietic and non-hematopoietic cells, whereas MIP-3β / CCL19 is expressed in thymus, lymph node, appendix, tonsil, as well as in mature DC, monocytes and lymphocytes 5. However, whether or not neutrophils produce MIP-3α / CCL20 and MIP-3β / CCL19 has never been investigated. Herein, we show that, following stimulation with either LPS or TNF-α, neutrophils are able to synthesize and release biologically active MIP-3α / CCL20 and MIP-3β / CCL19.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Espression of MIP-3α / CCL20 and MIP-3β / CCL19 mRNA in neutrophils and PBMC

To investigate whether MIP-3α / CCL20 and MIP-3β / CCL19 mRNA are expressed in neutrophils, the latter were cultured with either 100 ng / ml LPS or 5 ng / ml TNF-α for Northern blot analysis. Fig. 1 shows that resting neutrophils did not express any detectable MIP-3α / CCL20 or MIP-3β / CCL19 mRNA, while LPS- or TNF-α-treated cells exhibited a considerable accumulation of chemokine transcripts. Interestingly, kinetics of MIP-3α / CCL20 mRNA expression differed greatly from those of MIP-3β / CCL19 mRNA in stimulated PMN, as MIP-3α / CCL20 transcripts reached a maximum at 1 h and then rapidly declined, whereas MIP-3β / CCL19 mRNA accumulated at maximal levels at 21 h (Fig. 1, panels A and B). Other stimuli previously shown to induce chemokine mRNA in neutrophils, such as fMLP and GM-CSF 1, 2, failed to affect MIP-3α / CCL20 and MIP-3β / CCL19 steady-state mRNA levels (data not shown). Consistent with the genuine ability of stimulated neutrophils to express MIP-3α / CCL20 and MIP-3β / CCL19 mRNA were additional Northern blot experiments in which, in accord with previous reports 15, 16, IL-6 mRNA was expressed only in LPS-stimulated autologous PBMC (Fig. 1 C). Thus, the inducible expression of the MIP-3α / β genes in PMN is not likely to derive from the low numbers of contaminating PBMC in our PMN suspensions.

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Figure 1. Expression of MIP-3α / CCL20 and MIP-3β / CCL19 mRNA in human neutrophils. Total RNA was extracted from neutrophils stimulated for the times indicated with either 100 ng / ml LPS (A), or 5 ng / ml TNF-α (B). In panel C, purified populations of PMN and PBMC isolated from the same donor were cultured for 1 h with or without 100 ng / ml LPS. Northern blot analysis for MIP-3α / CCL20, MIP-3β / CCL19 and IL-6 mRNA expression was performed. Ten μg total RNA were loaded on each gel lane. The experiments depicted in this figure are representative of three (A), two (B) and three (C), respectively.

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2.2 MIP-3α / CCL20 and MIP-3β / CCL19 release by stimulated neutrophils

Subsequently, we examined whether human neutrophils could also produce the MIP-3α / CCL20 and MIP-3β / CCL19 proteins. In agreement with our Northern blot data, Fig. 2 A illustrates that both LPS and TNFα stimulated a time-dependent extracellular release of MIP-3α / CCL20, with a progressive accumulation of the protein up to 42 h (not shown). Dose-response experiments confirmed that 5 ng / ml of TNF-α and 100 ng / ml of LPS represented optimal concentrations (data not shown), LPS being consistently (in 10 independent experiments) more potent than TNF-α. Neutrophils released considerable amounts of MIP-3α / CCL20 also in response to IgG-opsonized yeasts (Y-IgG) (not shown). Furthermore, PMN treated with IL-10 were greatly inhibited (by 52 ± 6 %, n = 4) in their capacity to release MIP-3α / CCL20 in response to LPS for 21 h (Fig. 2 B), but not to TNF-α, similarly to what was previously observed with other chemokines 2. Importantly, additional experiments demonstrated that the neutrophil-derived MIP-3α / CCL20 is not attributable to the minimal contamination of the PMN population with eosinophils (data not shown), which have been recently shown to express MIP-3α / CCL20 mRNA 17.

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Figure 2. (A): Time course of MIP-3α / CCL20 release by LPS- and TNF-α-stimulated human neutrophils. Neutrophils were cultured for up to 21 h at 37 °C with 100 ng / ml LPS or 5 ng / ml TNF-α. Culture supernatants were harvested and then processed for MIP-3α / CCL20 determination. The asterisks represent significant differences between stimulated and resting PMN. (B): Effect of IL-10 on MIP-3α / CCL20 release by stimulated neutrophils. PMN were cultured for 21 h with either LPS or TNF-α, in the presence or absence of IL-10 (100 U / ml), before MIP-3α / CCL20 determination. The asterisks represent significant differences between LPS- and IL-10 plus LPS-treated PMN. For both panels, results are expressed as the mean ± SD of averaged duplicate determinations from at least four independent experiments.

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Measurable levels of MIP-3β / CCL19 were also detected in supernatants harvested from neutrophils treated with LPS or TNFα (Table 1). However, in contrast to MIP-3α /  CCL20, antigenic MIP-3β / CCL19 started to be released only after 21 h of stimulation, progressively accumulating into the supernatants for up to 42 h (Table 1). As in the case of MIP-3α / CCL20, IL-10 exerted an inhibitory effect on MIP-3β / CCL19 release by LPS-treated PMN (Table 1).

Table 1. Extracellular release of MIP-3β / CCL19 by human neutrophilsa)
 21 h42 h
  1. a) PMN (8 × 106 / ml) were stimulated as indicated in Sect. 4.1 for 21 and 42 h. MIP-3β / CCL19 was determined in the cell-free supernatants by a specific ELISA. Values are means ± SD (pg / ml) obtained from the number of experiments indicated in the parentheses. nd: not detectable.

Mediumnd (n = 4)nd (n = 4)
LPSnd (n = 4)913 ± 293 (n = 4)
TNF-αnd (n = 3)756 ± 262 (n = 3)
IL-10nd (n = 3)nd (n = 3)
IL-10 + LPSnd (n = 3)442 ±  56 (n = 3)

2.3 MIP-3α / CCL20 and MIP-3β / CCL19 release by PBMC

To further exclude any role of contaminating mononuclear cells present in our neutrophil preparations, we determined the ability of PBMC to release MIP-3α / CCL20 and MIP-3β / CCL19. For this purpose, in selected experiments PBMC were purified (in addition to neutrophils) and stimulated for 42 h with LPS. Under these conditions, PBMC produced, on a per cell basis, approximately 20-fold more MIP-3α / CCL20 than PMN, but little or no MIP-3β / CCL19 (Fig. 3), clearly indicating that the cellular origin of MIP-3α / CCL20 and MIP-3β / CCL19 in our neutrophil suspensions is unlikely to include any substantial contribution by contaminating monocytes or lymphocytes.

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Figure 3. Comparison of the ability of neutrophils and PBMC to release MIP-3α / CCL20 and MIP-3β / CCL19. PMN and PBMC isolated from the same donor were cultured for 42 h with 100 ng / ml LPS, before measuring MIP-3α / CCL20 and MIP-3β / CCL19 antigenic protein in their culture supernatants. The experiment depicted in this figure is representative of three.

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2.4 DC migration to neutrophil- and PBMC-derived supernatants

To ascertain whether neutrophil-derived MIP-3α / CCL20 and MIP-3β / CCL19 were biologically active, we tested the ability of concentrated supernatants derived from neutrophils to induce DC migration in a chemotaxis assay in vitro. As shown in Fig. 4 A, immature CD-34+-derived DC, that are known to express CCR6 5, 6, 18 readily migrated in response to recombinant MIP-3α / CCL20 and this response was almost completely (> 95 %) inhibited in the presence of specific MIP-3α / CCL20 neutralizing mAb. Supernatants harvested from neutrophils and PBMC treated with LPS also exerted a chemotactic activity on immature DC and this activity was almost completely blocked by the anti MIP-3α / CCL20 mAb, indicating that this chemokine was the main effector chemotactic protein released by neutrophils (and PBMC) under these experimental conditions. Isotype matched control antibodies were completely ineffective (data not shown). In agreement with the RNA and protein data, supernatants harvested from resting leukocytes were ineffective (Fig. 4 A).

Since previous studies have shown that MIP-3β / CCL19 is active on mature CCR7-positive DC 6, 18, migration of mature DC was also evaluated. Fig. 4 B shows that LPS-stimulated neutrophils (but not control cells) released into the supernatant a chemotactic activity for mature (CD83+, CD86+, CD25+) DC. This activity was partially blocked (60 %) by neutralizing anti-MIP-3β / CCL19 specific polyclonal Ab, whereas isotype-matched control Ab were ineffective (data not shown). It is noteworthy that under the same experimental conditions, the anti-MIP-3β / CCL19 polyclonal Ab inhibited 97 % of DC migration to recombinant MIP-3β / CCL19. Conversely, supernatants obtained from resting and stimulated PBMC were devoid of any chemotactic activity (Fig. 4 B). The latter findings are in agreement with the data obtained at the level of protein release (Fig. 3).

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Figure 4. Effect of neutrophil-derived supernatants on DC migration. Chemotaxis of DC was performed in Boyden chambers with neutrophil / PBMC-derived supernatants and 12 nM recombinant chemokines, in the presence or absence of the indicated antibodies. The incubation time for chemotaxis was 1.5 h. Panel (A) shows the migration of immature DC, whereas panel (B) illustrates the migration of LPS-mature DC in response to concentrated neutrophil- and undiluted PBMC-derived supernatants, prepared after cell stimulation with 100 ng / ml of LPS for 42 h. Values are expressed as the mean number ± SD of migrated cells in five high-power fields (100 X).

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2.5 Rapid integrin-dependent adhesion activities induced by neutrophil-derived supernatants

Subsequently, we determined whether neutrophil-derived supernatants could trigger rapid lymphocyte adhesion to purified integrin ligands, a recently reported biological function of some chemokines 19. For this purpose, we used the mouse pre-B cell line, L1 / 2, stably transfected with the CCR6 cDNA, and murine lymphocytes purified from peripheral lymph nodes and Peyer's patches, which show high CCR7 expression and responsiveness to MIP-3β / CCL19 (20 and our unpublished data). As shown in Fig. 5, both recombinant MIP-3α / CCL20 and MIP-3β / CCL19 triggered rapid integrin-dependent adhesion of CCR6-transfected or murine lymphocytes to, respectively, VCAM-1 and ICAM-1 and such activities were markedly prevented by the respective neutralizing Ab whereas isotype matched control Ab were completely ineffective. Notably, MIP-3α / CCL20-, and MIP-3β / CCL19-induced rapid adhesion of CCR6-transfected or murine lymphocytes was a transient phenomenon and was maximal after 3 min. The latter is consistent with the transient nature of rapid integrin triggering under physiological conditions 21. No binding was induced in absence of agonists (data not shown). Concentrated supernatants from LPS-stimulated PMN induced rapid adhesion in either CCR6-transfected or murine lymphocytes to VCAM-1 and ICAM-1, respectively (Fig. 5). In contrast, no detectable binding was triggered by supernatants from resting cells (Fig. 5). Reminiscent of observations made with our chemotaxis assays, the adhesion triggered by the neutrophil-derived supernatants was substantially prevented by the neutralizing anti-MIP-3α / CCL20 and MIP-3β / CCL19 Ab (Fig. 5), with an average inhibition of approximately 75 % for MIP-3α / CCL20 and 70 % for MIP-3β / CCL19. In contrast, isotype-matched control Ab were completely ineffective.

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Figure 5. Neutrophil-derived supernatants trigger rapid integrin-dependent lymphocyte adhesion to purified integrin ligands. Mouse CCR6-transfected L1 / 2 cells (A) and normal murine lymphocytes (B) were challenged with concentrated neutrophil-derived supernatants and 0.5 μM recombinant chemokines, in the presence or absence of the indicated antibodies. Cells were allowed to adhere to slides coated with VCAM-1 (A), or ICAM-1 (B), as described in Sect. 4. Values are mean counts ± SD of adherent cells derived from at least three independent experiments for each condition.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This work demonstrates that human neutrophils are able to express and release MIP-3α / CCL20 and MIP-3β / CCL19 in vitro, when appropriately activated. MIP-3α / CCL20 and MIP-3β / CCL19 are two structurally related CC chemokines that have been suggested to play a fundamental role in DC trafficking to mucosal surfaces and lymphoid organs 22. However, they exert also chemotactic activities for other leukocyte types: memory T cells and B lymphocytes in the case of MIP-3α / CCL20 7, 8, B, T and activated NK cells for MIP-3β / CCL19 8, 11 – 14. As a result, our data raises the interesting possibility that neutrophils might play a heretofore unsuspected role in modulating the migration of the above cell types.

In our study, production of MIP-3α / CCL20 and MIP-3β / CCL19 in substantial amounts by human neutrophils were observed in cells stimulated with either TNF-α or LPS, the latter representing the most effective agonist. Importantly, we also provided evidence which clearly excludes the possibility that the results obtained in neutrophils with respect to MIP-3α / CCL20 and MIP-3β / CCL19 mRNA expression and release could be attributed to a contamination with PBMC or eosinophils. Finally, IL-10 was also found to strongly diminish the release of MIP-3α / CCL20 andMIP-3β / CCL19 in PMN stimulated with LPS.

Kinetics of MIP-3α / CCL20 mRNA steady-state levels in LPS- or TNF-α-treated PMN were faster than those of MIP-3β / CCL19 mRNA. In addition, MIP-3α / CCL20 was consistently secreted at significant levels much earlier than MIP-3β / CCL19. These patterns of neutrophil-derived MIP-3α / CCL20 and MIP-3β / CCL19 seem to correlate with the current kinetic model of DC activation / maturation 18. Indeed, it has been shown that immature DC, having a high expression of CCR6 6, once pick up antigen or are activated by LPS, TNF-αor CD40L, down-regulate CCR6, start to mature and express CCR7. Strikingly, this series of events seems to occur over a time course 18, 23 that closely resembles the kinetics of MIP-3α / CCL20 and MIP-3β / CCL19 release by neutrophils. In this context, we show that supernatants harvested from stimulated PMN exert chemotactic activities on mature and immature DC. These effects were almost completely neutralized by anti-MIP-3α / CCL20 and anti-MIP-3β / CCL19 antibodies, consistent with the presence of biologically active MIP-3α / CCL20 and MIP-3β / CCL19 in leukocyte-derived supernatants.

In addition, we provide evidence suggesting a role for neutrophil-derived MIP-3α / CCL20 and MIP-3β / CCL19 in tissue targeting of lymphocytes. Indeed, we demonstrate that supernatantsfrom stimulated neutrophils trigger rapid integrin-dependent adhesion of CCR6-transfected lymphocytes and primary murine lymphocytes to purified VCAM-1 and ICAM-1, respectively, in agreement with the ability to recombinant MIP-3α / CCL20 and MIP-3β / CCL19 to fully trigger lymphocyte adhesion to immobilized integrin ligands 19. The data clearly show that neutrophils release biologically active chemokines that could be potentially able to direct memory and naive lymphocyte trafficking. Together with the previously demonstrated pro-adhesive role of neutrophil-derived CXCR3 ligands MIG and IP-10 3, our data support the concept that lymphocyte tageting to, and microenviromental positioning inside, the inflamed tissues could be controlled,at least in their early steps, by chemokines released in situ by activated neutrophils.

In conclusion, based on the findings reported herein, we postulate that neutrophil-derived MIP-3α / CCL20 and MIP-3β / CCL19 might contribute to the diapedesis and recruitment of DC, at various stages of maturation, and immunocompetent T lymphocytes to sites of inflammation and disease. In the light of the differential expression kinetics of PMN-derived MIP-3α / CCL20 and MIP-3β / CCL19 chemokines, our data could support a model of neutrophil-mediated regulation of "regional immunity". Considering that neutrophils are usually the first cells to arrive to a site of inflammation, an infectious pathogen, for instance, could promote the release of LPS or TNF-α in the epithelial or mucosal tissue, and the subsequent production of neutrophil-derived MIP-3α /CCL20. The latter chemokine, in turn, could recruit at the inflammatory sites immature DC and CCR6high memory lymphocytes, thus leading to a prompt pathogen recognition and defence. As MIP-3β / CCL19 (together with SLC / 6Ckine) is primarily involved in the regulation of normal trafficking of mature DC, CCR7-positive naive T lymphocytes and B cells to lymphoid T zones 24, it is difficult to speculate that neutrophil-derived MIP-3β / CCL19 could significantly contribute to such events. However, an excessive production of MIP-3β / CCL19 (but not 6Ckine / SLC) is also observed "peripherically" and is supposed to be responsible for the formation of novel lymphoid structures that are typical of chronic inflammatory diseases such as rheumatoid arthritis 25, 26. One could therefore postulate that a local production of MIP-3β / CCL19 by activated neutrophils and other cell types 25 might prevent the migration of mature DC to lymph nodes and recruit B cells and effector / memory CCR7+ T lymphocytes 13 towards those sites of chronic inflammation that will develop into ectopic lymphoid tissue. Altogether, our findings might uncover a novel, unexpected role of neutrophils in orchestrating, via the release of MIP-3α / CCL20 and MIP-3β / CCL19, the recruitment of DC and lymphocytes, and therefore in contributing to direct the transition from innate resistance and adaptive immunity.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Purification and cultivation of PMN and PBMC

Highly purified granulocytes (> 98.5 %) and PBMC were isolated under endotoxin-free conditions from buffy coats of healthy donors, as previously described 27. The granulocyte populations contained usually < 4 % eosinophils (n = 25), as revealed by May-Grünwald-Giemsa staining. In selected experiments, granulocytes were depleted of eosinophilsaccording to the method described by Koenderman et al. 28. Immediately after purification, leukocytes were suspended in RPMI-1640 medium (Biowhittaker, Caravaggio, Italy) supplemented with 10 % low-endotoxin FCS (< 0.009 ng / ml, Seromed, Biochrom KG, Berlin, Germany), usually plated at 8 × 106 / ml in polystyrene flasks (Biowhittaker), and then cultured at 37 °C, 5 % CO2 atmosphere. Cells were routinely stimulated with 100 ng / ml LPS (from E. coli, serotype 026 : B6, purchased from Sigma, St. Lous, MO) or 5 ng / ml TNFα (Peprotech, Rocky Hill, NJ). In selected experiments, neutrophils were also stimulated with 10 nM fMLP (Sigma), 10 ng / ml GM-CSF (Peprotech), or heat-killed yeast particles opsonized with IgG (Y-IgG) at a particle / cell ratio of 2 : 1. In other experiments, neutrophils were pretreated for 15 min with 100 U / ml IL-10 (kindly provided by Dr. K. Moore, DNAX, Palo Alto, CA), before stimulation with either LPS or TNF-α. At the indicated times, cell-free supernatants were harvested and stored at − 20 °C, whereas cell pellets were extracted for total RNA. All reagents used were of the highest available grade and were dissolved in pyrogen-free water for clinical use 27.

4.2 Preparation of DC

DC were generated as previously described 18. DC were also prepared from purified cord blood CD34+ cells and cultured as previously described 29.Mature DC were obtained after culturing in the presence of 100 ng / ml LPS for 24 h.

4.3 Isolation of murine lymphocytes

Peripheral lymph nodes (axillary, brachial, cervical and subiliac) and Peyer's patches were isolated from young adult BALB / c mice (Harlan, Udine, Italy) and gently pressed through a stainless fine wire steel mesh 30. Pooled lymphocytes (approximately 40 % B lymphocytes and 60 % T lymphocytes) were then washed with RPMI 1640 contain-ing 5 % FCS and resuspended in RPMI supplemented with 1 mM sodium pyruvate, 1 mM glutamine, 5 × 10− 3 M 2-mercaptoethanol, and 10 % FCS. For the in vitro adhesion assays, lymphocytes were resuspended in PBS, 1 mM CaCl2, 1 mM MgCl2, 10 % FCS, pH 7.2.

4.4 RNA isolation and Northern blot analysis

Total RNA from PMN and PBMC was extracted and analyzed as already described 27. Filters were hybridized using MIP-3α / CCL20, MIP-3β / CCL19, IL-6 and actin 15 cDNA fragments 32P-labeled using a Ready-to-go DNA labelling kit (Pharmacia, Uppsala, Sweden). MIP-3α / CCL20 and MIP-3β / CCL19 cDNA fragments were prepared by R. Bonecchi (Mario Negri Institute) by RT-PCR of mRNA purified from stimulated human DC. Oligonucleotide primers designed to obtain MIP-3α / CCL20 cDNA were: sense 5′-ATGT-GCTGTACCAAGAGTTTGC-3prime; and antisense 5′-TTACAT-GTTCTTGACTTTTTTACTGAGGAG-3prime;. Oligonucleotide primers designed to obtain MIP-3β / CCL19 were: sense 5′-TGGCACCAATGATGCTGAAGACTG-3prime; and antisense 5′-GTCATAGGTTAACTGCTGCGGCG-3prime;.

4.5 MIP-3α / CCL20 and MIP-3β / CCL19 antigenic determination

Antigenic MIP-3α / CCL20 and MIP-3β / CCL19 were measured in the cell-free supernatants by specific ELISA developed with specific antibodies purchased from R & D Systems (Minneapolis, MN), using the protocol previously described 31. The ELISA Amplification System (Life Technologies, Inc. Rockville, MD) was used to enhance the sensitivity of only MIP-3β / CCL19 detection. The MIP-3α / CCL20 ELISA had a detection limit of 10 pg / ml and did not cross-react with 3.2 ng / ml MIP-3β / CCL19, 3.2 ng / ml SLC / CCL21 and 3.2 ng / ml MIP-1β / CCL4. The MIP-3β / CCL19 ELISA had a detection limit of 200 pg / ml and did not cross react with and 3.2 ng / ml MIP-3α / CCL20, 3.2 ng / ml SLC / CCL21 and 3.2 ng / ml MIP-1β / CCL4.

4.6 Migration assay

Cell migration was evaluated using a chemotaxis chamber (Neuroprobe), Pleasanton, CA) and polycarbonate filters (5 μm pore size, Neuroprobe) as previously described 18. Cell suspensions (0.7 × 106 – 1 × 106 / ml) were incubated at 37 °C for 90 min. Leukocyte-derived supernatants or recombinant chemokines (12 nM) were also preincubated at 37 °Cfor 30 min with 15 μg / ml of anti-human MIP-3α / CCL20 (MAB360, R & D), 5 μg / ml goat anti-human MIP-3β / CCL19 (#500-P50, Peprotech), or matched control antobidies, to neutralizetheir chemotactic activities. Each experiment was performed in triplicates. PBMC-derived supernatants from resting or LPS-treated cells were used undiluted, whereas supernatants from resting or stimulated neutrophils usually needed to be concentrated approximately 100-fold prior to use (using Centricon Plus 20 devices from Amicon Inc., Beverly, MA 3. Spontaneous migration was determined in the absence of supernatants.

4.7 Adhesion assay

To evaluate the presence of a proadhesive activity in neutrophil-derived supernatants, we used the mouse pre-B lymphocyte cell line L1 / 2 stably transfected with human CCR6 cDNA (kindly provided by Dr. M.E. Fuentes, Roche Biosciences, Palo Alto, CA), and murine lymphocytes (see above). Eighteen-well glass slides were precoated for 16 – 20 h at 4 °C with 20 μl of purified native mouse VCAM-1 or ICAM-1, diluted under the critical micellar concentration in PBS. The adhesion assay was performed as previously described 3, 32. Neutrophil-derivedsupernatants were the same as those used for the chemotaxis assays. Adherent cells in 0.2 mm2 were calculated by computer-assisted enumeration, by using the image analysis software NIH-Image 1.62. Each experiment was performed in quadruplicate for each condition.

4.8 Statistical analysis

Values are expressed as means ± SD and analyzed by Student's t-test for paired data. p values < 0.05 were considered significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

We thank Federica Calzetti for excellent technical assistance and Walter Luini (Mario Negri Institute, Milano, Italy) for performing, with invaluable skill, the chemotaxis experiments. We would like to thank Dr. P.P. McDonald for his criticisms and suggestions. This work was supported by grants from MURST (COFIN and 60 %), Fondazione Cassa di Risparmio VR-VI-BL-AN "Progetto Sanià", and "Consorzio per lo Studio e lo Sviluppo degli Studi Universitari di Verona". Patrizia Scapini was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC).

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    Power, C. A., Church, D. J., Meyer, A., Alouani, S., Proudfoot, A. E., Clark-Lewis, I., Sozzani, S., Mantovani, A. and Wells, T. N., Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3α from lung dendritic cells. J. Exp. Med. 1997. 186: 825 – 835.
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    Sozzani, S., Allavena, P., D'Amico, G., Luini, W., Bianchi, G., Kataura, M., Imai, T., Yoshie, O., Bonecchi, R. and Mantovani, A., Differential regulation of chemokine receptors during dendritic cell maturation. A model for their trafficking properties. J. Immunol. 1998. 161: 1083 – 1086.
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    Campbell, J. J., Bowman, E. P., Murphy, K., Youngman, K. R., Siani, M. A., Thompson, D. A., Wu, L., Zlotnik, A. and Butcher, E. C., 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3β receptor CCR7. J. Cell Biol. 1998. 141: 1053 – 1059.
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    Sozzani, S., Allavena, P., Vecchi, A. and Mantovani, A., The role of chemokines in the regulation of dendritic cell trafficking. J. Leuk. Biol. 1999. 66: 1 – 9.
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    Cyster, J. G., Chemokines and cell migration in secondary lymphoid organs. Science 1999. 286: 2098 – 2102.
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    Sallusto, F. and Lanzavecchia, A., mobilizing dentritic cells for tolerance, priming, and chronic inflammation. J. Exp. Med. 1999. 189: 611 – 614.
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  • 27
    Cassatella, M. A., Bazzoni, F., Flynn, R. M., Dusi, S., Trinchieri, G. and Rossi, F., Molecular basis of IFNγ and LPS enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components. J. Biol. Chem. 1990. 265: 20241 – 20246.
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  • 29
    Sozzani, S., Longoni, D., Bonecchi, R., Luini, W., Bersani, L., D'Amico, G., Borsatti, A., Bussolino, F., Allavena, P. and Mantovani, A., Human monocyte-derived and CD34 + cell-derived dendritic cells express functional receptors for platelet activating factor. FEBS Lett. 1997. 418: 98 – 100.
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  • 31
    Scapini, P., Calzetti, F. and Cassatella, M. A., On the detection of neutrophil-derived vascular endothelial growth factor (VEGF). J. Immunol. Methods 1999. 232: 121 – 129.
  • 32
    Laudanna, C., Campbell, J. J. and Butcher, E. C., Role of Rho in chemoattractant-activated leukocyte adhesion through integrins. Science 1996. 271: 981 – 983.