Published online before print January 3, 2005
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Department of Microbiology and Immunology, University of South Alabama, Mobile
1 Correspondence: Department of Microbiology and Immunology, MSB 2096, University of South Alabama, Mobile, AL 36607. E-mail: rlausch{at}jaguar1.usouthal.edu
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(Mig). Purified mouse neutrophils were stimulated directly by IFN- to secrete these chemokines, and neutrophils at the DTH site expressed IP-10. IFN- knockout mice, which manifested depressed ear-swelling following DTH challenge, made little IP-10 and no Mig. Reconstitution of these mice with IFN- induced CXCR3 ligand synthesis. Depletion of neutrophils or CD4+ T cells but not CD8+ T cells markedly reduced IFN- levels, suggesting the former were direct (or indirect) cellular sources of this cytokine. Collectively, our results support the hypothesis that neutrophil production of T cell-recruiting chemokines contributes to the regulation and amplification of the DTH response.
Key Words: T lymphocyte
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Neutrophils from man and mouse are now recognized as one cellular source of chemokines in inflammatory responses, including those triggered by virus infection [3
, 9
, 10
]. Moreover, human neutrophils have been shown to secrete the lymphocyte-recruiting chemokines interferon-inducible protein 10 [(IP-10)/CXC10] and monokine induced by IFN- [(Mig)/CXCL9], which share the receptor CXCR3 [11
, 12
]. IP-10 and Mig have been shown specifically to be involved in cellular recruitment to inflammatory sites such as the central nervous system and brain after infection with mouse hepatitis virus [13
, 14
], the liver during adenovirus and murine cytomegalovirus infection [15
, 16
], and at the site of various skin diseases [17
].
In this study, we test the hypothesis that the neutrophil is a crucial cell linking the innate and acquired immune responses through the production of chemokines, which are essential for the recruitment of effector T cells to the site of inflammation. Given the association of CXCR3 but not CCR5 with DTH, we hypothesize that neutrophils rapidly recruited to the DTH site are induced to produce the lymphocyte-recruiting chemokines IP-10 and Mig. The expression of these chemokines would be expected to facilitate recruitment of CXCR3-expressing cells, including CD4+ T cells, and further amplify the inflammatory cascade. To investigate this hypothesis, neutrophils were examined for production of IP-10 at the DTH site, and the effect of neutrophil depletion on lymphocyte recruitment and chemokine production was assessed. We also examined whether IFN- was required for the generation of IP-10 and Mig at the DTH site.
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gene knockout (GKO) mice (BALB/c background) were obtained from Jackson Laboratories (Bar Harbor, ME). All animals were cared for according to federal, state, and local regulations.
Antibodies and reagents
Recombinant mouse IFN- and interleukin (IL)-1
were purchased from R&D Systems, Inc. (Minneapolis, MN). The rat hybridoma RB6 8C5, which produces anti-mouse granulocyte monoclonal antibody (mAb), was a gift from Robert Coffman (DNAX Research Institute, Palo Alto, CA). This antibody was prepared as described previously [18
]. Rat hybridoma clones GK1.5 (anti-CD4) and 2.43 (anti-CD8) were obtained from the American Type Culture Collection (Manassas, VA). RB6 8C5, preconjugated to phycoerythrin (PE; PharMingen, San Diego, CA), rabbit anti-CXCR3 antibody (a gift from Dr. Robert Strieter, University of California School of Medicine, Los Angeles), and a hamster anti-IP-10 mAb (a gift from Dr. Andrew Luster, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown) were used for immunohistochemistry. Rat, rabbit, and hamster immunoglobulin G (IgG) and appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).
Induction of DTH
Mice were sensitized with 1 x 104 plaque-forming units (PFU)/2 µl HSV-1 strain RE on the scarified right cornea. As described previously [19
], the DTH response was monitored using the ear-swelling assay. Ten days postinfection, 10 µl RPMI-1640 medium + 5% newborn calf serum (NCS) containing 1 x 105 PFU HSV-1 RE was injected into the right ear pinna using a 30-gauge needle and Hamilton syringe (Hamilton, Reno, NV.) The left ear received a control dose of medium only. Twenty-four hours later, ear-swelling was measured using a Mitutoyo 7326 micrometer (Schlessinger Tools, New York, NY). This time-point was identified previously as corresponding to the maximal DTH response against HSV-1 in our model [3
]. Results were expressed as the mean swelling of the virus-challenged ear minus the mean swelling of the control ear (units 10–4 inches).
Cellular depletion
Mice were depleted of neutrophils, CD4+ T cells, or CD8+ T cells by intraperitoneal (i.p.) injection of 1 mg RB6 8C5, 0.5 mg GK1.5, or 1 mg 2.43 antibody, respectively, 24 h prior to DTH challenge in the ear. Control mice received i.p. injection of rat IgG. Depletion of neutrophils was confirmed by differential staining (HEMA3®, Biochemical Sciences, Inc., Swedesboro, NJ) of blood smears to be, on average, >84%. CD4+ and CD8+ cellular depletions were confirmed by fluorescence-activated cell sorter analysis of spleen cells to be, on average, >89% and >92%, respectively.
Immunohistological staining
Mice were sensitized and depleted of neutrophils as described above. Control mice received rat IgG. Twenty-four hours postchallenge, ear tissue was removed from the DTH site, and 30 µm cryosections were prepared as described previously [3
]. Sections were stained for the presence of neutrophils (rat mAb, RB6 8C5), CD4+ T cells (rat mAb, GK1.5), and CXCR3+ cells (rabbit anti-CXCR3). A total of 24 fields was counted from ear pinna sections from each experimental group using a fluorescent microscopy at x40 magnification. Cell infiltration was quantitated by counting in a coded manner, and the reader was unaware of the treatment given. Dual staining of neutrophils (RB6 8C5 preconjugated to PE) and IP-10 (hamster anti-IP-10 primary and anti-hamster-FITC secondary) was performed on 5 µm cryosections from the DTH site. Appropriate isotype controls were included. Sections were examined with the 40x oil immersion objective of a confocal microscope. To demonstrate colocalization of neutrophil and IP-10-specific antibodies,the PE and FITC channel images were merged.
Neutrophil isolation and stimulation
Neutrophils were isolated from the bone marrow of mice and purified to a level of >99%, as described previously [3
]. For in vitro stimulation, 106 neutrophils were plated in triplicate in 24-well tissue-culture plates, which had been precoated with NCS. Stimulation was performed with medium alone (RPMI 1640+5% NCS) or containing recombinant mouse IFN- or recombinant mouse IL-1 at 1 and 10 ng/ml. Cells were stimulated for 8 h at 37°C in 5% CO2, after which time, supernatants were removed and assayed for chemokine production using enzyme-linked immunosorbent assay (ELISA).
IFN and chemokine assay
Ear sections (5 mm2) from the DTH site were excised at 24 h postchallenge and were placed in 500 µl RPMI 1640 + 5% NCS. Tissues were homogenized, sonicated for 30 s, and clarified by centrifugation at 150 g for 10 min. The resultant tissue lysates were assayed for IP-10, Mig, or IFN- by ELISA (R&D Systems, Inc.). The level of sensitivity for each ELISA kit was 2.2 pg/ml, 3 pg/ml, and 2 pg/ml, respectively.
IFN reconstitution
Mice were sensitized as above. Recombinant mouse IFN- (500 ng) was admixed with 10 µl virus challenge dose (1x105 PFU HSV-1 RE) and injected into the ear pinna of GKO mice. This dose was determined to result in a level of IFN- >500 pg/5 mm2 section of ear pinna after 24 h.
Statistical analysis
Student’s t-test was performed to determine the significance between DTH responses and chemokine production between control and test groups within an individual experiment. The level of confidence for significance was P < 0.05. Experiments were performed at least two to three times with four to five mice per group. A representative experiment is shown in each figure.
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Figure 1. Effect of neutrophil depletion on lymphocyte recruitment to the DTH site. Mice sensitized to HSV-1 were treated with mAb RB6 8C5 to deplete neutrophils. Controls received rat IgG. Naive mice were included for comparison and represent a primary infection. The mice were challenged in the ear pinna with HSV-1, and 24 h later, tissue samples were removed and cut for immunohistological staining. Infiltrating neutrophils and CD4+ and CD8+ cells were detected using mAb RB6 8C5, GK1.5, and 2.43, respectively. CXCR3+ cells were stained using a rabbit anti-CXCR3 polyclonal antibody. Cell numbers were quantitated in 24 fields per group, at x40 original magnification, and the mean number of cells per field is shown.
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Figure 2. Effect of neutrophil depletion on IP-10 and Mig production at the DTH site. Neutrophils were depleted by i.p. injection of RB6 8C5 mAb 1 day prior to viral challenge in mice sensitized to HSV-1. Control mice were given rat IgG. Mice (n=4) were challenged in the ear pinna with 1 x 105 PFU HSV-1, and 24 h later, ear pinna lysates were prepared. Levels of IP-10 and Mig were quantitated by ELISA. A naive group is shown for comparison and represents a primary infection with an equivalent dose of HSV-1.
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-stimulated neutrophils secrete IP-10 and Mig in vitro or IL-1 for 8 h. Culture supernatants were then assayed by ELISA for IP-10 and Mig. Figure 3
shows that the neutrophils secreted substantial levels of both chemokines in a dose-dependent manner when stimulated with IFN-. It is interesting that the early-warning cytokine IL-1 did not induce neutrophils to make either chemokine. In addition, we observed that neutrophils exposed to infectious HSV-1 [multiplicity of infection (MOI)=1] in vitro produced high levels of IP-10 (>2100 pg/ml±41) but little or no Mig (>7.5 pg/ml±7).
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Figure 3. Purified neutrophils secrete IP-10 and Mig in response to IFN-. Neutrophils were purified from bone marrow. Neutrophils (106) were stimulated in triplicate with 1 or 10 ng/ml IL-1 or IFN-. Control samples were not stimulated. After 8 h of incubation at 37°C, supernatants were collected and assayed by ELISA for IP-10 or Mig production.
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Figure 4. Neutrophils at the DTH site stain positive for IP-10 protein. Wild-type (WT) mice sensitized to HSV-1 were challenged in the ear pinna with 1 x 105 PFU HSV-1. Twenty-four hours later, 5 mm2 sections of ear were excised from the DTH site and embedded for sectioning. Sections (5 µm) were stained simultaneously with the anti-neutrophil antibody RB6 8C5, preconjugated with PE and a hamster anti-IP-10 mAb or appropriate isotype controls. A secondary anti-hamster-FITC antibody was used to visualize IP-10 staining. All antibodies were used at a concentration of 1 µg/ml, and representative images are shown. (A) Anti-IP-10 staining; (B) anti-neutrophil staining; (C) merged image for colocalization of the two antibodies; (D) PE isotype control; (E) FITC control.
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is required for the production of IP-10 and Mig at the DTH site is a potent inducer of IP-10 and Mig in vitro. To test whether this cytokine played a role in the production of these ligands at the DTH site, we conducted DTH experiments in IFN- knockout mice (GKO). It was found that ear swelling in the GKO mice was reduced by 53% when compared with the WT controls (Fig. 5A
). In addition, little or no IP-10 or Mig could be detected in the ear pinnas of the GKO hosts (Fig. 5B)
. However, administration of 500 ng IFN-, locally at the time of HSV-1 challenge, restored the hosts’ ability to make both chemokines, suggesting that IFN- is required for their production during DTH.
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Figure 5. IFN- is required for optimal DTH responsiveness and chemokine production. IFN- GKO mice and WT mice (n=4) were sensitized on the scarified cornea with 1 x 104 PFU HSV-1. Ten days later, mice were challenge in the ear pinna with 1 x 105 PFU HSV-1. DTH measurements were taken 24 h later (A). GKO mice were injected with 500 ng recombinant mouse IFN- admixed with the virus-challenge dose into the ear pinna. Twenty-four hours postchallenge, chemokine levels were assessed by ELISA (B).
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? can act at the DTH site to stimulate production of IP-10 and Mig, we investigated whether neutrophils were a cellular source of this proinflammatory cytokine. Figure 6
shows two representative experiments in which IFN- production was reduced by 78% (Exp. 1) and 74% (Exp. 2) at the DTH site in mice depleted of neutrophils. However, when purified neutrophils were stimulated in vitro with 10 ng/ml IL-1 or tumor necrosis factor (TNF-), alone or in combination, no IFN- could be detected (data not shown). Phorbol 12-myristate 13-acetate (30 ng/ml) and HSV-1 (MOI 1–10) also failed to induce IFN- secretion (data not shown), although all agonists tested readily induced neutrophils to make the chemokine MIP-2. These results suggested that neutrophils are most likely involved indirectly in IFN- production.
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Figure 6. Neutrophil depletion suppresses IFN- levels at the DTH site. Sensitized mice (n=4) were treated with mAb RB6 8C5 (neutrophil-depleted group) or rat IgG (control group). These two groups plus naïve hosts were challenge with HSV-1, and 24 h later, ear pinna lysates were assayed for IFN- by ELISA. Two independent experiments are shown.
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and chemokine expression at the DTH site. We therefore evaluated whether cell depletion of one or both of these subsets affected IFN-, IP-10, and/or Mig levels at the DTH site. As observed in our previous studies [3
], depletion of CD4+ T cells markedly reduced ear swelling (66% reduction), and CD8+ T cell depletion had no significant effect. Figure 7
shows that CD4+ T cell depletion had a dramatic effect on cytokine and chemokine expression, producing an 88% reduction in IFN-, a 65% reduction in IP-10, and an 85% reduction in Mig relative to the IgG-treated controls. In marked contrast, CD8+ T cell depletion did not alter IFN- or chemokine levels significantly.
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Figure 7. Effect of T lymphocyte subset depletion on production of IFN-, IP-10, and Mig at the DTH site. Sensitized mice were depleted of CD4+ or CD8+ T cell subsets by i.p. injection of the relevant antibody as described in Materials and Methods. Control mice received rat IgG. Mice were challenged in the ear pinna with 1 x 105 PFU HSV-1 the following day. Twenty-four hours postchallenge, the DTH response was assessed via ear swelling measurement. Then, 5 mm2 tissue sections were excised, and lysates were prepared for IFN- or chemokine measurement using ELISA.
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could be induced to secrete both of these ligands. This suggested that neutrophils may be a direct cellular source of these chemokines at the DTH site. It is interesting that infectious HSV-1 was able to stimulate neutrophils to produce high levels of IP-10 but little Mig, and IL-1 failed to stimulate neutrophils to make IP-10 or Mig, although this early-warning cytokine can readily induce these cells to make other CXC molecules as well as CC chemokines [3
]. IP-10 and Mig have also been reported to be produced by human neutrophils exposed to IFN-, provided a costimulator such as TNF- or lipopolysaccharide was also present [9
, 12
]. Additional support for neutrophils as a direct source of CXCR3 binding chemokines in vivo is shown by the colocalization of an anti-IP-10 antibody with neutrophils in tissue from the DTH site. It is evident from Figure 4
that not all neutrophils at the DTH site stain positive for the production of IP-10. This may reflect neutrophils that have ceased to secrete IP-10 or have not yet been stimulated to produce the chemokine. Although in vivo depletion of neutrophils reduced the levels of IP-10 and Mig significantly at the inflammatory site, chemokine production was not abrogated completely. This result may reflect the inability to completely deplete neutrophils in vivo (depletions of 80–90% were achieved by RB6 8C5 mAb treatment, as judged by immunohistochemical staining of cellular infiltrates in the ear pinna). Alternatively, other cellular sources of IP-10 and Mig may be present in the ear pinna during DTH. For example, dermal fibroblasts have been demonstrated to be an important source of IP-10 in the skin and plasma during atopic dermatitis [23 ], and tissue-resident dendritic cells have been shown to produce IP-10 and Mig in vitro [24 , 25 ]. We have also observed that tissue from uninfected mouse ear pinnas constitutively expresses low levels of IP-10 protein (S. J. Molesworth-Kenyon and R. N. Lausch unpublished data).
To explore the role of IFN- in vivo, we conducted experiments in mice with a targeted disruption of this cytokine gene. In agreement with the observations of Bouley et al. [26
], mice lacking IFN- displayed a reduced DTH response to HSV-1 antigen. We found that these animals also manifested a marked reduction in IP-10 and Mig at the DTH site. Local transfer of IFN- restored Mig and IP-10 expression. The work of McLoughlin et al. [27
] has shown that an IFN- dose, >84-fold lower than that used in our study, was capable of restoring neutrophil infiltration into the inflammatory site in GKO mice. However, IFN- reconstitution did not fully restore the DTH swelling response in our model (data not shown). One reason for this may be that a single administration of exogenous IFN- did not reconstitute chemokine levels to those observed in WT mice, and such levels may be required for robust ear-swelling. In addition, it is known that mice lacking IFN- have an impaired, primary immune response to HSV-1 [26
, 28
], presumably as a result of insufficient generation of primed TH1 T cells.
IFN- is the predominant cytokine associated with TH1-mediated immune responses. Thus, it was not surprising that depletion of CD4+ T cells produced a striking reduction in this cytokine at the DTH site. This suggests that CD4+ T cells were a major source of this cytokine following antigen challenge. IP-10 and Mig levels were also markedly reduced. Their reduction probably reflects the loss of IFN- as an inducer, although CD4+ T cells may also secrete these chemokines. Depletion of CD8+ T cells did not significantly alter IFN-, IP-10, or Mig levels. These latter results are consistent with our earlier studies [3
] and contrast with results in contact hypersensitivity models in which CD8+ T cells were identified as a major, direct source of IFN- [29
, 30
] and an indirect source of IP-10 [29
, 31
].
IFN- levels were also sharply reduced in ear pinna lysates of neutrophil-depleted hosts. The most logical explanation for this is that removal of neutrophils resulted in decreased CXCR3 ligand expression and thereby less recruitment of IFN--producing T cells. Alternatively, neutrophils may produce IFN- directly [32
]. In the past, these phagocytes have not been associated with IFN- synthesis. Recently, however, a number of investigators have reported that neutrophils can make IFN- following exposure to selected infectious agents [33
34
35
36
37
]. In our hands, neutrophils exposed to infectious HSV-1 were not stimulated to produce IFN- (unpublished observation). Whether the cytokine milieu at the DTH site has this potential remains to be determined. If so, the IFN- released by the neutrophil could act in an autocrine manner to induce IP-10 and Mig secretion. An alternative cellular source of IFN- might be the natural killer (NK) cell, which is reported to accumulate at the site of HSV-1 infection [38
, 39
] and play a critical role in innate protection against HSV-2 infection [40
].
Collectively, our results are compatible with the following scenario: NK cells recruited to the DTH site, early on in the inflammatory response, act as an initial source of IFN- to stimulate infiltrating neutrophils to secrete CXCR3 ligands. These chemokines in turn could recruit CD4+ T cells. The subsequent infiltration of CD4+ T cells would contribute to the further production of IFN- and result in increasing IP-10 and Mig secretion by newly arriving neutrophils. In such a way, the inflammatory cascade at the DTH site would be amplified by the progressive infiltration of NK, neutrophil, and CD4+ T cells.
In conclusion, we have demonstrated a novel role for the neutrophil as a source of lymphocyte-recruiting chemokines during DTH-to-HSV-1 antigen. By producing IP-10 and Mig, the neutrophil influences the nature and robustness of the acquired immune response. Our results may have relevance to other inflammatory models in which neutrophils have been implicated in the recruitment of T lymphocytes [41 42 43 44 45 ].
Received September 1, 2004; revised October 25, 2004; accepted December 7, 2004.
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