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Infection and Immunity, January 2003, p. 524-526, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.524-526.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Production of Macrophage Inflammatory Protein 3{alpha} (MIP-3{alpha}) (CCL20) and MIP-3ß (CCL19) by Human Peripheral Blood Neutrophils in Response to Microbial Pathogens

Tohru Akahoshi,1* Takeshi Sasahara,2 Rie Namai,3 Toshimichi Matsui,3 Hiroyuki Watabe,3 Hidero Kitasato,2 Matsuhisa Inoue,2 and Hirobumi Kondo3

Departments of Laboratory Medicine,1 Microbiology,2 Internal Medicine, Kitasato University School of Medicine, Kanagawa, Japan3

Received 11 July 2002/ Returned for modification 26 August 2002/ Accepted 4 October 2002


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ABSTRACT
 
Effects of bacterial pathogens on the production of macrophage inflammatory protein 3{alpha} (MIP-3{alpha}) and MIP-3ß from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.


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TEXT
 
Neutrophils are the principal cells of host defense against microbial pathogens. Neutrophils are the first cells to arrive at the site of inflammation and play a crucial role in innate immunity (11). Antigen-specific immune responses are also involved in bacterial infection, followed by neutrophil-mediated innate immune responses. Migration of T lymphocytes and dendritic cells (DC) to the inflammatory sites occurred with a time lag following the accumulation of neutrophils.

Infiltration of inflammatory cells to the sites of bacterial infection is caused by endogenous production of various chemoattractants. Chemokines are recently identified chemotactic cytokines and can be divided into four subfamilies, CXC, CC, CX3C, and C (16). Novel members of the CC chemokines, macrophage inflammatory protein 3{alpha} (MIP-3{alpha}) (CCL20) and MIP-3ß (CCL19), and their specific receptors, CCR6 and CCR7, have been recently identified (5, 7, 13, 15). MIP-3{alpha} is chemotactic for memory T cells and immature DC, and MIP-3ß is chemotactic for naïve and activated T lymphocytes and mature DC. Since antigen presentation of DC to T cells is an essential process for acquired immune responses, production of these chemokines may be important for host defense against bacterial pathogens (3). It has recently been demonstrated that neutrophils are capable of producing MIP-3{alpha} and MIP-3ß in response to lipopolysaccharide (LPS) and proinflammatory cytokines (9). However, it is not known whether neutrophils produce these chemokines through direct interaction with bacteria. In this study, we evaluated the effects of microbial pathogens on the production of MIP-3{alpha} and MIP-3ß from neutrophils.

Neutrophils were incubated with killed bacteria at a ratio of 1:10 (neutrophils to bacteria) at 37°C using RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum. Killed bacteria were prepared by treatment with 0.6% formalin for 2 h. Neutrophils were obtained from healthy volunteers and the purity was >97%. Gene expression was determined by reverse transcription-PCR followed by Southern blot hybridization. The primers used for MIP-3{alpha} were 5'-TTGGATCCTGCTGCTACTCCACCTCTG-3' and 5'-TTCTCGAGTATATTTCACCCAAGTCTGTTTT-3' and for MIP-3ß were 5'-TTGGATCCTCCCAGCCTCACATCACTCACACC-3' and 5'-TTCTCGAGTAACTGCTGCGGCGCTTCATCTT-3' (7). Southern blot hybridization was performed by using digoxigenin end-labeled probes (MIP-3{alpha}, 5'-GATGTCACAGCCTTCATTGG-3'; MIP-3ß, 5'-AGTCTCTGGATGATGCGTTCTACC-3'). As shown in Fig. 1A, neutrophils cultured with medium alone did not express detectable amounts of either chemokine mRNA, while Escherichia coli rapidly increased the expression of MIP-3{alpha} as early as 30 min after exposure. Gene expression of MIP-3{alpha} reached a maximum level at 0.5 to 2 h and persisted up to 20 h. The microbes also induced the gene expression of MIP-3ß in neutrophils, although it was first detected at 6 h and increased in time, with the maximal expression reached at 20 h. Neutrophils were exposed to gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) or gram-negative bacteria (E. coli, Salmonella enterica serovar Typhimurium, and Pseudomonas aeruginosa) at a ratio of 1:10 for 6 h and the gene expressions were determined. Both gram-positive and gram-negative bacteria enhanced MIP-3{alpha} and MIP-3ß expression in neutrophils (Fig. 1B). Neutrophils were also exposed to viable bacteria (E. coli or S. aureus) at a ratio of 1:1 for 2 h at 37 °C and then incubated for the indicated periods in the presence of antibiotics. Viable bacteria also enhanced gene expression of MIP-3{alpha} and MIP-3ß in neutrophils, and the induction kinetics were the same as those of killed bacteria (data not shown).



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  		FIG. 1. Induction of MIP-3{alpha} and MIP-3ß in neutrophils by microbial pathogens. (A) Neutrophils were incubated with killed E. coli at a ratio of 1:10 for the indicated times using RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum. (B) Neutrophils were incubated with gram-positive or gram-negative bacteria at a ratio of 1:10 for 6 h. Gene expression of MIP-3{alpha} and MIP-3ß was determined by reverse transcription-PCR followed by Southern blot hybridization. These results were representative of three separate experiments using neutrophils isolated from different donors.

To identify chemokine expression, neutrophils were incubated with E. coli at a ratio of 1:10 and fixed with 4% paraformaldehyde. Immunoperoxidase staining was carried out using a Dako (Carpinteria, Calif.) Envision/horseradish peroxidase-3,3'-diaminobenzidine tetrachloride kit. Mouse antibodies against MIP-3{alpha} and MIP-3ß (Genzyme Techne, Cambridge, Mass.) and biotinylated goat anti-mouse immunoglobulin G were used. Polymorphonuclear neutrophils apparently expressed MIP-3{alpha} at 24 h (Fig. 2A) and MIP-3ß at 48 h (Fig. 2B). Control cells incubated with medium alone did not express detectable amounts of these chemokines.



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  		FIG. 2. Immunostaining of MIP-3{alpha} and MIP-3ß in neutrophils. Neutrophils were incubated with E. coli at a ratio of 1:10 and fixed with 4% paraformaldehyde. Immunostaining of MIP-3{alpha} (A) and MIP-3ß (B) was performed by using mouse antibodies against human MIP-3{alpha} and MIP-3ß.

Production of chemokines by E. coli-stimulated neutrophils was determined by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn.). As shown in Fig. 3A, E. coli significantly increased the generation of both chemokines in a time-dependent manner. Release of MIP-3{alpha} was detected as early as 12 h and increased thereafter, whereas MIP-3ß production was first found at 48 h after the stimulation. Neutrophils were cocultured with various microbial pathogens (1:10) or LPS (10 ng/ml) for 48 h and chemokine production was determined. Both gram-positive and gram-negative bacteria significantly enhanced the generation of MIP-3{alpha} from neutrophils. Concentrations of MIP-3{alpha} in the culture supernatants of bacteria-stimulated neutrophils were almost the same as those in supernatants of LPS-stimulated neutrophils. MIP-3ß was also detected, although it was secreted in a lesser amount than MIP-3{alpha} (Fig. 3B).



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  		FIG. 3. Production of chemokines by neutrophils stimulated with microbial pathogens. (A) Neutrophils were incubated with killed E. coli at a ratio of 1:10 for the indicated times. (B) Neutrophils were incubated with various microbial pathogens (1:10) or LPS (10 ng/ml) for 48 h. Chemokines in the culture supernatant were determined by enzyme-linked immunosorbent assay. Data represent means ± standard deviations. These results were representative of three separate experiments using neutrophils isolated from independent donors.

A number of studies have demonstrated that neutrophils are capable of producing various chemokines, including those from the CXC subfamily (interleukin-8, growth-related gene product [GRO{alpha}], gamma interferon [IFN-{gamma}]-inducible protein 10 [IP-10], monokine induced by IFN-{gamma} [MIG], and IFN-inducible T-cell {alpha} chemoattractant protein [I-TAC]) and the CC subfamily (MIP-1{alpha} and MIP-1ß), in response to various inflammatory stimuli (10). Interleukin-8 and GRO{alpha} are potent chemoattractants for neutrophils and basophils (6), and IP-10, MIG, and I-TAC can promote T-cell migration (1, 4). Neutrophil-derived CC chemokines MIP-1{alpha} and MIP-1ß are chemotactic factors for macrophages, T cells, and DC. Therefore, elaboration of these chemokines from activated neutrophils may cause the subsequent migration of various leukocyte subsets to the inflamed sites.

DC are professional antigen-presenting cells derived from hematopoietic stem cells. DC are target cells for MIP-3{alpha} and MIP-3ß, because immature DC express CCR6 and mature DC express CCR7 (2, 12). It has also been shown that naïve T cells express CCR7 and memory T cells express both CCR6 and CCR7 (8). Thus, MIP-3{alpha} and MIP-3ß are chemokines that potentially promote selective recruitment of DC and T-cell subsets. Furthermore, Yang et al. demonstrated that the neutrophil-derived antibiotic peptide ß-defensin was selectively chemotactic for CCR6-expressing DC and T cells (14). These findings indicate that increased production of MIP-3{alpha}, MIP-3ß, and ß-defensin by neutrophils may initiate antigen-specific immune responses by recruiting DC and T cells to the site of bacterial invasion.

Scapini et al. recently demonstrated that neutrophils could generate MIP-3{alpha} and MIP-3ß in response to LPS and tumor necrosis factor alpha (9). We here reported that direct interaction of neutrophils with either gram-positive or gram-negative bacteria could stimulate the production of these chemokines. Recognition of the bacterial components and products or phagocytosis of bacteria by neutrophils may evoke stimulatory signals to generate various chemokines, including MIP-3{alpha} and MIP-3ß. Overall, accumulation of neutrophils in bacterial infections may contribute to not only innate immune responses but also acquired immune responses against bacterial pathogens through the generation of various chemokines, including MIP-3{alpha} and MIP-3ß.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Laboratory Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. Phone: 81-42-778-9119. Fax: 81-42-778-8441. E-mail: akahoshi{at}med.kitasato-u.ac.jp. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, January 2003, p. 524-526, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.524-526.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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