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The Journal of Immunology, 2008, 180, 510 -521
Copyright © 2008 by The American Association of Immunologists, Inc.
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Multichannel Fluorescence Spinning Disk Microscopy Reveals Early Endogenous CD4 T Cell Recruitment in Contact Sensitivity via Complement1

M. Ursula Norman*,{dagger}, Sara Hulliger*, Pina Colarusso* and Paul Kubes2,*

* Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Alberta, Canada; and {dagger} Centre for Inflammatory Disease, Department of Medicine, Monash University, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Contact sensitivity (CS) is one of the primary in vivo models of T cell-mediated inflammation. The presence of CS-initiating CD4 T lymphocytes at the time of challenge is essential for transfer and full development of the late phase CS inflammatory response. From this observation investigators have speculated that early recruitment of CD4 T cells to the site of challenge must occur. Moreover, there must be rapid synthesis/release and disappearance of an important mediator during the first hours after hapten challenge. Using spinning disk confocal microscopy, we observed the very early effector events of the immune response. Simultaneous, real-time visualization of predominant neutrophil and extremely rare CD4 T cell trafficking in the challenged skin vasculature was noted (one rolling CD4 T cell for every 10–18 rolling and adherent neutrophils). We demonstrate that neutrophil adhesion during the early CS response was reduced in C5a receptor-deficient (C5aR–/–) mice or leukotriene B4 receptor antagonist-treated mice, whereas CD4 T cell recruitment was only inhibited in C5aR–/– mice. In line with these observations, leukocyte infiltration and the associated tissue damage were significantly reduced in C5aR–/– mice but not in leukotriene B4 receptor antagonist-treated wild-type mice 24 h after challenge. C5a receptor expression on T cells and not on tissue resident cells was important for the development of a CS response. Thus, by using spinning disk confocal microscopy we visualized the early events of an adaptive immune response and identified the rare but essential recruitment of CD4 T cells via the complement pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Contact sensitivity (CS)3 is an inflammatory skin response that serves as a classical model of T cell-mediated immunity. During CS, an initial sensitization period is induced following cutaneous exposure to a chemical hapten. Subsequent exposure of the skin to the offending hapten results in substantial leukocyte infiltration into tissue, edema formation, and significant tissue injury (reviewed in Ref. 1). This elicitation phase can be further subdivided into an early phase occurring within the first 2 h of challenge and a late inflammatory phase, which peaks between 8 and 24 h (2, 3). The early phase is characterized by the activation of the complement system (4, 5, 6, 7), mast cell degranulation, and the release of vasoactive proinflammatory mediators such as histamine, serotonin, and TNF-{alpha} leading to neutrophil recruitment (8, 9, 10). Indirect evidence suggests that during this time point a small population of T cells is recruited to the challenged site to initiate the late phase CS response. Indeed, the 24-h CS response is transferred to CD4 T cell-deficient mice if these mice are treated with sensitized CD4 T cells from wild-type mice at the time of challenge but not when transferred just 2 h after hapten challenge (2).

Based on these data we hypothesized that a small population of CD4 T cells are recruited to the challenged skin site in response to the rapid synthesis/release of an important mediator during the first 2 h after hapten challenge. However, direct evidence that CD4 T cells enter the skin site within the first 2 h has yet to be provided. Moreover, the identity and function of the chemoattractant(s) responsible for this early lymphocyte recruitment in the first 2 h of CS in mice remains to be fully elucidated. Because the window in which CD4 T cells can transfer CS is within the first 2 h, the mediator must be very short lived. Although most emphasis regarding lymphocyte recruitment has been placed on a family of chemotactic cytokines, namely chemokines, temporal evidence would not support a role for this family of molecules. For example, the expression of chemokines at the site of hapten challenge is slightly elevated over baseline levels at 2 h after hapten challenge, with persistent expression throughout the CS response that reaches peak levels at 24 h (5). As such, this profile of chemokine expression is not consistent with a short 2-h window of opportunity to initiate CD4 T cell recruitment.

The early CS phase coincides with early complement activation by IgM-Ag complexes and the local elaboration of C5a (4, 5, 6, 7, 11). Indeed, the production of C5a has been shown to be critical to the development of the CS response. Both anti-C5 treatment and complement blockade have been shown to significantly inhibit the late phase 24 h CS response, as evidenced by reduced ear swelling, cell infiltration, and impaired chemotactic activity (11). Although the mechanism of C5a action remains unclear, ear extracts from sensitized mice suggested chemotactic activity that was C5a dependent and was limited to very early time points. Indeed, inhibition of C5a 3 h after Ag challenge no longer reduced the 24-h late phase response (5). As such, C5a was hypothesized to activate local C5a receptor-bearing cells such as mast cells to release inflammatory mediators that cause endothelial cell activation and indirectly recruit effector cells into the skin (11, 12, 13). C5a has also been proposed to interact with C5a receptors on endothelial cells, enhancing vascular permeability and adhesion molecule expression (14, 15). However, with constitutive expression of adhesion molecules such as E- and P-selectin in the skin, this mechanism may have less importance in this vascular bed (16, 17, 18). In addition, C5a might act directly as a lymphocyte chemoattractant during the contact sensitivity response, because C5a receptor expression has been shown on T lymphocytes (19, 20). A limiting factor to test these possibilities is that most techniques, including histology or standard intravital microscopy, are not sufficiently sensitive to detect and/or track the very few endogenous effector CD4 T cells that might infiltrate the tissue at early time points.

It should be noted that in some studies C5a deficiency did not completely inhibit the development of a CS response (11). As such, another rapidly produced nonchemokine chemotactic molecule is leukotriene B4 (LTB4), which has been shown to be important in the early recruitment of neutrophils in inflammation. Recent studies have shown that LTB4 can also act as a T lymphocyte chemoattractant. LTB4, through its BLT1 receptor, has been shown to mediate the firm adhesion and chemotaxis of effector T cells in vitro (21, 22, 23). LTB4 has also been shown to be responsible for the early accumulation of effector T cells to sites of inflammation in an in vivo model of asthma (23). Interestingly, LTB4 is rapidly produced within the skin at very early time points during the CS response, with an early peak at 1 h after hapten challenge (24). Thus, these findings make LTB4 an attractive target as a potential mediator of leukocyte recruitment during the early phase of CS. In the current study we used spinning disk confocal microscopy to track multiple populations of leukocytes in real time and identified for the first time an infrequent but rapid influx of CD4 T cells into the skin microvasculature in CS. Although both C5a and LTB4 inhibition blocked early neutrophil recruitment into skin, only C5a mediated the early lymphocyte recruitment and inhibited the late phase leukocyte recruitment and tissue damage associated with CS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice were purchased from The Jackson Laboratory. C5a receptor-deficient (C5aR–/–) mice on a C57BL/6 background were a gift from Dr. C. Gerard (Harvard Medical School, Boston, MA). Mice were maintained in a specific pathogen-free, double-barrier unit at the University of Calgary (Calgary, Alberta, Canada). The protocols used were in accordance with the guidelines drafted by the University of Calgary Animal Care Committee and the Canadian Council on the Use of Laboratory Animals. Mice were used between 6 and 10 wk of age.

Oxazolone-induced CS in the flank skin

Mice were sensitized by the topical application of 50 µl of 5% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich) in an acetone-olive oil vehicle (4:1) to the shaved flank. Six or 7 days later, mice received a 50-µl challenge of 1% oxazolone solution to the upper right abdomen of the mouse. As a control, some sensitized mice received a 50-µl challenge of the acetone/olive oil vehicle solution. Skin venules were visualized via intravital microscopy at various time points after Ag challenge. At the conclusion of the experiments, a sample of inflamed skin was taken to estimate tissue swelling/edema and dried over 24 h (values shown as a percentage of tissue swelling: [(wet weight – dry weight)/(dry weight)]).

To confirm the results seen in the flank skin, some experiments were repeated in the ear. Mice were sensitized as described above and 6 or 7 days later mice received a 10-µl challenge of 1% oxazolone solution on the ventral aspect of the left ear. The thickness of Ag-challenged ears was measured using an Engineer’s dial micrometer (Mitutoyu). Ear thickness was calculated by subtracting the thickness of the unchallenged right ear from that of the challenged left ear.

Histology

Tissue samples were fixed in 10% formalin, processed, and H&E stained by the staff in the Department of Histopathology at the University of Calgary. In addition, esterase staining was performed as previously described (25). Slides were stained for granulocytes with a chloroacetate esterase (Leder) stain (Sigma-Aldrich) and analyzed by light microscopy in a blinded fashion. Leukocyte numbers were determined by counting the number of positive-stained cells over 10 fields at a magnification of x400.

Intravital microscopy

Leukocyte-endothelium interactions were studied in the microcirculation of mouse flank skin. Animals were anesthetized by i.p. injection of a mixture of 10 mg/kg xylazine hydrochloride (MTC Pharmaceuticals) and 200 mg/kg ketamine hydrochloride (Rogar/STB). The right jugular vein was cannulated to administer additional anesthetic and fluorescent dyes.

The microcirculation of the ventral abdominal skin was prepared for microscopy as previously described (26). Briefly, a midline abdominal incision was made extending from the pelvic region up to the level of the clavicle. The skin was separated from the underlying tissue, remaining attached laterally to ensure that the blood supply remained intact. The area of skin was then extended over a viewing pedestal and secured along the edges using 4.0 sutures. The loose connective tissue lying on top of the dermal microvasculature was carefully removed by dissection under an operating microscope. The exposed microvasculature was immersed in isotonic saline and covered with a coverslip held in place with vacuum grease. To visualize endogenous leukocytes, animals were injected with 50 µl of 0.05% (i.v.) rhodamine 6G (Sigma-Aldrich). Fluorescence was visualized by epi-illumination using 510 and 560 filters.

The skin microvasculature was visualized using an intravital microscope (Axioskop; Carl Zeiss) with a x40 water immersion objective lens (Weltzlar; E. Leitz) with a x10 eyepiece. A video camera (Panasonic 5100 HS) was used to project images on to a monitor and the images were recorded for playback analysis using a videocassette recorder. Three to six dermal venules (20- to 40-µm in diameter) were selected in each experiment.

The numbers of rolling and adherent leukocytes were determined off-line during video playback analysis. Rolling leukocytes were defined as those cells moving at a velocity slower than that of the erythrocytes within a vessel. Leukocyte rolling flux was determined by counting the number of leukocytes that rolled by a fixed point in the venule during 1 min. Leukocyte rolling velocity was determined by measuring the time required for a leukocyte to roll along a 100-µm length of venule. Rolling velocity was determined for 20 leukocytes at each time interval. Leukocytes were considered adherent to the venular endothelium if they remained stationary for 30 s or longer.

Spinning disk confocal microscopy

The microcirculation of the ventral abdominal skin was prepared for microscopy as described above. The flank skin microvasculature was visualized using a spinning disk confocal microscope. Images were acquired with an Olympus BX51 upright microscope using a x20/0.95 XLUM Plan Fl water immersion objective. The microscope was equipped with a confocal light path (WaveFx; Quorum) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation). Anti-CD4-FITC (L3T4; BD Biosciences; 3 µg/mouse) and anti-mouse Ly-6G PE (Gr-1; BD Biosciences; 2 µg/mouse) were injected i.v. into wild-type and C5aR–/– mice to image CD4+ T lymphocytes and neutrophils, respectively. Both 488- and 561-nm laser excitation wavelengths (Cobalt, Stockholm, Sweden) were used in rapid succession and visualized with the appropriate long-pass filters (Semrock). Typical exposure time for both excitation wavelengths was 168 ms. A 512 x 512 pixel back-thinned electron-multiplying charge-coupled device camera (C9100-13, Hamamatsu) was used for fluorescence detection. Volocity Acquisition software (Improvision) was used to drive the confocal microscope.

Simultaneous CD4 T lymphocyte and neutrophil rolling and adhesion were assessed in 20 random fields of view in the postcapillary venules of the skin.

LTB4 blockade studies

Zileuton was administered (3 mg/kg (27) in 2% DMSO and 0.9% saline solution; Cayman Chemicals) to mice i.v. 15–30 min before challenge with oxazolone. For 24-h CS experiments, zileuton was administered a total of three times every 2 h from the initial injection. Control animals received an equivalent volume of the vehicle solution (2% DMSO and 0.9% saline). This dosing regimen has been reported to prevent leukotriene synthesis for a minimum of 24 h (28).

The specific LTB4 receptor (BLT1) antagonist CP-105,696 ((+)-1-(3S,4R)-[3-(4-phenylbenzl)-4-hydroxychroman-7-yl]-cyclopentane carboxylic acid) was a gift from Pfizer Groton. CP-105,696 was administered to mice i.p. (3 mg/kg in DMSO and 0.9% saline solution) 30 min before challenge with oxazolone (29).

Adoptive transfer experiments

Wild-type or C5aR–/– mice sensitized with oxazolone were killed on day 4 and spleen and peripheral lymph node cells were isolated. After RBC lysis, isolated cells were fluorescently labeled with CFSE (5 µM) for 20 min at room temperature. Mononuclear cells (5 x 107) were administered i.v. to unsensitized wild-type or C5aR–/– mice before oxazolone challenge (1% oxazolone). Some mice that received sensitized CFSE-labeled wild-type cells were pretreated with the LTB4 receptor antagonist (CP105,696) before oxazolone challenge. As a control, some animals received an i.v. injection of saline before oxazolone challenge. Endogenous leukocyte-endothelial (rhodamine-labeled) interactions as well as CFSE-labeled mononuclear-endothelial interactions were examined by intravital microscopy 2 and 24 h after hapten challenge. CFSE-labeled mononuclear cell adhesion was assessed over 40–50 fields of view. Several images of the skin were recorded using a camera attached to an Olympus 1X70 microscope (x100 magnification) to assess the number of transferred CFSE-labeled mononuclear cells that entered the extravascular tissue.

Statistics

All data are displayed as mean ± SEM. Data were analyzed using standard statistical analysis (ANOVA and Student’s t test with Bonferroni’s correction for multiple comparison where appropriate). Statistical significance was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Contact hypersensitivity response in mouse flank skin is biphasic

Although intravital microscopy of the mouse ear circulation allows the observation of leukocyte trafficking without surgical manipulation during an inflammatory response, tissue swelling decreases visibility at later time points (30). To avoid this complication in the current study, we observed leukocyte-endothelial interactions in the dermal microvasculature of the mouse flank. Intravital microscopy of the mouse flank skin revealed that following challenge with oxazolone, leukocyte rolling velocity was significantly decreased from baseline levels at all time points tested, which is suggestive of increased adhesiveness of the vascular endothelium (Fig. 1A). By comparison, vehicle treatment (olive oil/acetone) did not alter leukocyte rolling velocity (Fig. 1A). Oxazolone treatment induced a significant decrease in leukocyte rolling flux at the 2-, 4-, 8-, and 24-h time points from baseline levels (data not shown). This decrease in flux is due to the significant drop in rolling velocity, allowing cells to accumulate in the venules. Indeed, the number of rolling cells seen in the venule at any time increased over the levels seen in vehicle-treated mice (Fig. 1B).


Figure 1
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  		FIGURE 1. A–C, Leukocyte rolling velocity (A), the number of rolling leukocytes per 100 µm (B), and leukocyte adhesion (C) in flank skin postcapillary venules of vehicle (olive oil/acetone)-treated and oxazolone-treated C57BL/6 mice are shown. Leukocyte-endothelial interactions were examined before hapten challenge (0 h), during the early phase after hapten challenge (2–3 h), and during the late phase CS response (4, 8, and 24 h). D, Tissue edema of flank skin from untreated (No Challenge), vehicle-treated, and oxazolone-treated (Oxa) mice at 2 and 24 h after hapten challenge is shown. E, Total number of leukocyte infiltration per field of view (fov) in the skin of unchallenged, 2-h CS, and 24-h CS C57BL/6 mice. F, Differential leukocyte counts were performed on esterase-stained histology sections under x400 magnification. The numbers of mast cells (MC), granulocytes (Gran), lymphocytes (Lymph), and monocytes/macrophages (Mn/Mq) per field of view were determined. Data are shown as mean ± SEM of 6–12 animals (*, p < 0.05 relative to vehicle at indicated time points; **, p < 0.05 relative to no challenge group; #, p < 0.05 relative to vehicle 0-h time point).

 
Although leukocyte adhesion was significantly increased from baseline by oxazolone challenge at all time points tested, leukocyte adhesion appeared to follow a biphasic pattern (Fig. 1C). A small peak of leukocyte adhesion was induced at 2 h after hapten challenge. This was reduced at 3 h followed by a larger peak of adhesion starting at 4 h and extending to 24 h after hapten challenge. The adhesion response at 24 h was significantly enhanced compared with levels seen at 2 h. The acetone/olive oil vehicle is a known irritant and challenge with this mixture also increased adhesion, although only within the first 2 h and at a lower level than that seen in the oxazolone group (Fig. 1C). Thus, the vehicle causes a very minor response compared with the hapten-specific response. Leukocyte recruitment within the first 2 h of CS in the postcapillary venules of skin was dependent on P- and E-selectin (data not shown), while the late phase recruitment was selectin independent (data not shown). This pattern of recruitment is identical with that observed during the CS response in the mouse ear (2).

Although leukocyte rolling and adhesion as assessed by intravital microscopy revealed differences between vehicle and oxazolone-treated mice 2 h after hapten challenge, standard intravital microscopy was not sufficiently sensitive to discriminate between the different types of rolling leukocytes that were recruited (data not shown). Moreover, no differences between treatments were observed by using tissue swelling as a measurement of early tissue injury in skin flank (Fig. 1D). Tissue swelling was dramatically increased from the levels seen in vehicle-treated mice 24 h after oxazolone challenge. Thus, while tissue swelling is adequate to assess the late phase inflammatory response in the flank skin, it is not sufficiently sensitive to examine the early CS response.

To determine the number of adherent cells that entered the tissue in wild-type mice during the CS response, we examined H&E- and esterase-stained tissue sections of the mouse flank. Relatively few cells were found extravascularly in tissue sections taken from 2-h CS mice. The few cells that infiltrate the tissue at this time point do not significantly increase the total number of infiltrated leukocytes over the numbers seen in unchallenged mice (Fig. 1E). By contrast, at the 24-h CS time point a 10-fold increase in the number of infiltrated leukocytes was observed compared with the number at the 2-h CS time point. A systematic assessment of the tissues revealed a significant increase in the number of infiltrating granulocytes, lymphocytes, and a smaller number of monocytes (Fig. 1F).

C5a and LTB4 both mediate leukocyte adhesion during the early phase of CS

To determine the role of C5a and LTB4 in regulating the early leukocyte-endothelial interactions, we examined 2-h CS responses in the skin microvasculature of wild-type, C5aR–/–, and LTB4 antagonist-treated mice. Intravital microscopy revealed that leukocyte rolling parameters were not different between C5aR–/– and wild-type mice at all time points tested (data not shown). However, leukocyte adhesion was significantly diminished in C5aR–/– mice compared with that in wild-type mice during the early phase of the CS response (Fig. 2A). The very minor increase in tissue swelling in the skin observed in wild-type mice treated for 2 h with oxazolone was diminished in C5aR–/– mice (Fig. 2B).


Figure 2
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  		FIGURE 2. A, Leukocyte adhesion in flank skin postcapillary venules of untreated and oxazolone-treated C57BL/6 mice and C5aR–/– mice 2 h after challenge. B, Tissue swelling was assessed in the flank of C57BL/6 mice and C5aR–/– mice. C, Leukocyte adhesion in flank skin postcapillary venules of unchallenged, vehicle-, zileuton-, and CP105,696-treated C57BL/6 mice 2 h after hapten challenge. Data are shown as mean ± SEM of 6–12 animals (*, p < 0.05 relative to no challenge group; #, p < 0.05 relative to C57BL/6 paired time point).

 
To identify the potential role for leukotrienes in the early CS response, we first treated wild-type mice with zileuton, which inhibits the enzyme 5-lipoxygenase (5-LO). 5-LO catalyzes the first steps in leukotriene biosynthesis; thus, inhibiting 5-LO prevents the generation of a wide range of leukotrienes (LTB4, LTC4, LTD4, and LTE4). Pretreatment of wild-type mice with zileuton did not alter the number of rolling leukocytes at the 2-h time point from that seen in untreated mice (data not shown). However, pretreatment with zileuton resulted in a significant decrease in leukocyte adhesion compared with that in vehicle-treated mice at this time point (Fig. 2C). This indicates that a chemoattractant that is generated by the 5-LO pathway is important in leukocyte recruitment during the early phase of CS response. To identify the leukotriene responsible for the abrogation of leukocyte recruitment during the early CS response, the compound CP105,696 was used to specifically inhibit the LTB4 receptor (BLT1). CP105,696 pretreatment did not alter the number of rolling leukocytes from that seen in untreated mice at the 2-h time points (data not shown). However, CP105,696 treatment significantly inhibited leukocyte adhesion at the early CS time point (Fig. 2C). Edema in the skin was not affected if mice were pretreated with CP105,696 or zileuton (data not shown).

Chemoattractants can regulate both leukocyte adhesion in the vasculature and leukocyte emigration into the tissues, and the latter closely associates with tissue injury. We examined H&E- and esterase-stained tissue sections at the 2-h CS time point in wild-type, C5aR–/–, and LTB4 antagonist-treated mice. Tissue injury in wild-type, C5aR–/–, and LTB4 antagonist-treated mice was minimal at 2 h after hapten challenge with very little leukocyte infiltration or ulceration of the epidermis (data not shown). Our hypothesis was that only a very small number of lymphocytes need to be recruited during the early phase of the CS response to create an environment for delayed hypersensitivity. Because standard histology or intravital microscopy could not detect these cells, more sensitive approaches were needed to track the recruitment of lymphocytes vs other leukocyte populations at the early time point.

C5aR deficiency, not LTB4 inhibition, blocks early CD4 T cell recruitment

Standard intravital microscopy required manual switching between different filters to be able to distinguish individual cell types. This restricted our ability to simultaneously track different populations of leukocytes in real time. Indeed, it appeared by using this technique that the majority, if not all, leukocytes were neutrophils (2). Two-photon microscopy, although sufficiently sensitive to detect two distinct populations of leukocytes, is not capable of tracking rapid events like rolling cells in vivo. However, by using spinning disk confocal microscopy with rapid automatic filter switching we were able to track two different populations of leukocytes rolling and adhering in the skin microvasculature. Very small amounts of fluorescently labeled Abs (anti-mouse CD4-FITC and anti-mouse Gr-1 PE antibodies) were administered into wild-type and C5aR–/– mice to directly visualize the simultaneous trafficking patterns of endogenous CD4 T lymphocytes and neutrophils (Fig. 3, A and B). Approximately 10–18 rolling neutrophils for every rolling CD4 T cell were observed in wild-type and C5aR–/– mice (supplemental video files 1 and 2 and Fig. 3, C and D).4


Figure 3
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  		FIGURE 3. A and B, Simultaneous images of Gr-1+ neutrophils and CD4 T lymphocyte interactions in the postcapillary venules of C57BL/6 (A) and C5aR–/– (B) mice 2 h after hapten challenge. C and D, The total number of rolling neutrophils (C) and rolling CD4 T lymphocytes (D) were assessed over several fields of view in C57BL/6 and C5aR–/– mice 2 h after hapten challenge. E–H, The total number of adherent neutrophils (E and G) and adherent CD4 T lymphocytes (F and H) were assessed over several fields of view (fov) in C57BL/6 mice (E–H), C5aR–/– mice (E and F), and CP105,696-pretreated C57BL/6 mice (G and H). A minimum of three animals were completed at each time point (*, p < 0.05 relative to C57BL/6 mice).

 
The numbers of adherent neutrophils and CD4 T cells were significantly different in wild-type compared with C5aR–/– mice. Neutrophil adhesion per field of view was significantly decreased in C5aR–/– mice by ~60% compared with that in wild-type mice (Fig. 3E). More importantly, a striking reduction in the CD4 T lymphocyte adhesion per field in C5aR–/– mice was observed with a near abolition of arrested lymphocytes (Fig. 3F). Although it was not possible to distinguish whether the CD4 T cells were responsible for the neutrophil adhesion or vice versa, inhibition of the LTB4 receptor provides us with some insight. LTB4 receptor blockade inhibited neutrophil adhesion (Fig. 3G) but had no effect on CD4 T cell adhesion 2 h after hapten challenge (Fig. 3H). These data suggest that lymphocytes adhere independently from neutrophil adhesion. LTB4 receptor blockade did not impact neutrophil or lymphocyte rolling (data not shown).

C5aR on CD4 T cells is responsible for lymphocyte recruitment

Although we demonstrate herein that lack of the C5a receptor prevents CS-initiating CD4 T cell adhesion in dermal postcapillary venules in the first 2 h of CS, it is unclear whether this is due to direct effects on the lymphocytes or perhaps to inhibition of the C5a receptor on tissue resident cells (mast cells, endothelium) that could subsequently recruit CD4 T lymphocytes. Therefore, adoptive transfer experiments were next performed. Because both B and T cells are required to transfer the CS response to naive mice (1, 7, 31, 32), a mixed population of immune cells derived from the spleens and peripheral lymph nodes of either wild-type or C5aR–/–-sensitized mice were labeled with CFSE and transferred to naive mice before hapten challenge. The CS response generated by the adoptive transfer of these cells was assessed via intravital microscopy 2 h later. The number of transferred rolling CFSE-labeled mononuclear cells at the 2-h time point was the same regardless of whether the cells had a C5a receptor or not (Fig. 4A). Moreover, treatment with CP105,696, the LTB4 receptor antagonist, also did not affect the number of rolling CFSE-labeled wild-type cells in blood vessels (Fig. 4A). However, the number of adherent CFSE-labeled C5aR–/– cells was strikingly reduced compared with CFSE-labeled wild-type cells (Fig. 4B). In fact, the adhesion of CFSE-labeled wild-type cells in skin postcapillary venules was 5-fold greater than that of CFSE-labeled C5aR–/– cells. At this time point, most of the transferred fluorescent cells were present in the vasculature with a very small number that could be seen in the tissue, but only when wild-type cells were used (Fig. 4C). The numbers of extravascular cells were too low to be able to accurately quantitate this parameter. The CP105,696 treatment did not reduce the number of mononuclear cells that adhered in the skin postcapillary venules (Fig. 4B). It should be noted that despite having no effect on the CFSE-labeled mononuclear cell population, CP105,696 significantly decreased the level of endogenous leukocyte adhesion to basal levels at 2-h CS (data not shown) and, as already demonstrated, almost all of those leukocytes were neutrophils.


Figure 4
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  		FIGURE 4. A and B, CFSE-labeled wild-type and C5aR–/– mononuclear cell rolling flux (A) and adhesion (B) in the postcapillary venules of flank skin of naive wild-type mice with or without CP105,696 treatment 2 h after oxazolone challenge. fov, Field of view. C, Image of CFSE mononuclear wild-type cells present in the vasculature and extravascular tissue (arrow) 2 h after hapten challenge, visualized via an Olympus 1X70 microscope (original magnification, x100). A minimum of three animals was completed at each time point (*, p < 0.05 relative to wild-type cells).

 
C5aR-deficiency, not LTB4 inhibition, blocks delayed 24-h responses in tissue

Leukocyte emigration into the tissues at 24 h closely correlates with tissue injury. H&E- and esterase-stained tissue sections were examined at the 24-h CS time point in wild-type, C5aR–/– mice, and LTB4 antagonist-treated mice (Fig. 5). At 24 h after hapten challenge, wild-type mice had very profound tissue injury characterized by microabscess formation, leukocyte infiltration around hair follicles, and macroscopic ulceration of the epidermis compared with unchallenged or 2-h postchallenged mice (Fig. 5, A and B). All of these sequelae were noticeably reduced in C5aR–/– mice (Fig. 5C). At 24 h of CS, leukocyte infiltration was reduced by >60% in C5aR–/– mice compared with wild-type mice (Fig. 5D). There was a general reduction in all types of leukocytes, but only the number of lymphocytes reached significance (Fig. 5E). Total leukocyte recruitment at 24 h after oxazolone challenge was similar in mice receiving the 5-LO inhibitor (zileuton), the LTB4 receptor antagonist (CP105,696), or vehicle (Fig. 5F). These compounds did not alter the number and type of leukocyte infiltration (granulocytes, lymphocytes, or monocytes/macrophages) into the tissue at 24-h CS compared with vehicle treatment (Fig. 5G). Thus, although both C5a and LTB4 appear to block the early neutrophil adhesion to endothelium during CS, only the inhibition of C5a reduced the early lymphocyte recruitment, the late phase leukocyte infiltration into skin, and the subsequent tissue damage.


Figure 5
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  		FIGURE 5. A–C, Representative histological sections of flank skin taken from 2-h challenged (A) and 24-h challenged C57BL/6 mice (B) as well as 24-h challenged C5aR–/– mice (C). Sections were stained with H&E (original magnification, x100). Microabscess formation is indicated by the arrows. D and E, Total number of leukocyte infiltration into the skin of C57BL/6 and C5aR–/– mice 24 h after hapten challenge (D) and the differential leukocyte counts (E) that were performed on esterase stained histology sections (original magnification, x400). F and G, Total number of leukocyte infiltration into the skin of vehicle-, CP105,696-, and zileuton-treated C57BL/6 mice 24 h after hapten challenge (F) and the differential tissue leukocyte counts (G) that were performed on esterase-stained histology sections original magnification, x400). The numbers of mast cells (MC), granulocytes (Gran), lymphocytes (Lymph), and monocytes/macrophages (Mn/Mq) per field of view (fov) were determined. A minimum of 3–6 animals was completed at each experimental group (*, p < 0.05 relative to the C57BL/6 group).

 
Lymphocytes require C5aR to induce a complete CS response

Wild-type or C5aR–/– mononuclear cells were harvested, labeled with CFSE, injected into naive wild-type mice, and the late phase CS response was examined. Adoptive transfer of sensitized wild-type cells induced a robust 24-h CS response characterized by significantly increased granulocyte, lymphocyte, and monocyte/macrophage infiltration into tissue compared with that in hapten-challenged mice that did not receive a transfer of mononuclear cells (Fig. 6, A and B). However, cells derived from sensitized C5aR–/– mice were not able to initiate a 24-h CS response in naive wild-type mice after hapten challenge (Fig. 6, A and B). C5aR–/– mononuclear cell transfer did not result in any increase in granulocyte or lymphocyte infiltration. There was a slight but significant increase in monocyte/macrophage infiltration compared with hapten-challenged mice that received no cells (Fig. 6B).


Figure 6
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  		FIGURE 6. Endogenous leukocyte recruitment into the skin of wild-type mice in which CS was induced by the adoptive transfer of sensitized CFSE-labeled wild-type or C5aR–/– cells 24 h after hapten challenge into wild-type or C5aR–/– mice. The total number of endogenous leukocytes in the extravascular tissue (A and D) and the differential leukocyte counts (B and E) were determined from esterase-stained histology sections (original magnification, x400). The total number of infiltrating CFSE-labeled mononuclear cells (C and F) per field of view (fov) was determined from tissue sections using an Olympus 1X70 microscope (original magnification, x100). MC, Mast cells; Gran, granulocytes; Lymph, lymphocytes; Mn/Mq, monocytes/macrophages. A minimum of three animals were completed at each time point (*, p < 0.05 relative to no cells; #, p < 0.05 relative to wild-type cells group).

 
There was a very large increase in the number of CFSE-labeled wild-type mononuclear cells present in the extravascular tissue 24 h after hapten challenge. By comparison, there were very few CFSE-labeled C5aR–/– cells found in the skin 24 h after hapten challenge. Quantification of these data revealed an 8- to 10-fold increase in recruitment of CFSE-labeled wild-type cells compared with CFSE-labeled C5aR–/– cells (Fig. 6C). Pretreatment with CP105,696 did not alter the accumulation of sensitized CFSE-labeled wild-type cells in the extravascular tissue 24 h after oxazolone challenge (data not shown). In addition CP105,696 pretreatment did not alter the increase in endogenous leukocyte entry into the skin caused by the adoptive transfer of sensitized CFSE-labeled wild-type mononuclear cells to naive mice 24 h after oxazolone challenge (data not shown).

Interestingly, transferring sensitized CFSE-labeled wild-type cells into naive C5aR–/– mice also resulted in a robust 24-h CS response, with significantly enhanced endogenous leukocyte infiltration into the skin (Fig. 6D). In these experiments, the C5aR–/– mice did not exhibit any defect in the accumulation of granulocyte or mononuclear cells in response to oxazolone (Fig. 6E). In addition, there was no significant difference in the recruitment of CFSE-labeled wild-type mononuclear cells into the skin of C5aR–/– mice compared with that in wild-type mice (Fig. 6F), suggesting very little role for the C5a receptor on mast cells and endothelial cells during this response.

C5aR-bearing lymphocytes do not regulate late phase responses in the vasculature

Despite a very significant reduction in the leukocyte infiltration into tissues of actively sensitized C5aR–/– mice compared with that in wild-type mice 24 h after hapten challenge, leukocyte adhesion in the blood vessels of wild-type and C5aR–/– mice remained elevated at the 24-h time point (Fig. 7A). In fact, there was no significant difference in leukocyte adhesion between the two mouse strains at 24 h. Coincident with increased adhesion, there was increased tissue swelling in both wild-type and C5aR–/– mice at 24 h, suggesting that vascular inflammation persisted in both strains (Fig. 7B). These results were confirmed in ear skin, with similar ear swelling observed in the two mouse strains after 24 h of CS (Fig. 7C). Leukocyte adhesion (Fig. 7D) and edema formation (data not shown) 24 h after oxazolone challenge was similar in mice receiving the 5-LO inhibitor, the LTB4 receptor antagonist, or vehicle.


Figure 7
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  		FIGURE 7. A, Leukocyte adhesion in flank skin postcapillary venules of oxazolone-treated C57BL/6 mice and C5aR–/– mice 24 h postchallenge. B and C, Tissue swelling was assessed in the flank (B) and ears (C) of C57BL/6 mice and C5aR–/– mice. D, Leukocyte adhesion in flank skin postcapillary venules of unchallenged and vehicle-, zileuton-, and CP105,696-treated C57BL/6 mice 24 h after hapten challenge. Data are shown as mean ± SEM of 6–12 animals (*, p < 0.05 relative to the 2-h group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A biphasic distribution of leukocyte adhesion within the postcapillary venules of the flank skin was observed, with a small, transient peak of adhesion at 2 h followed by a larger persistent peak at 24 h after oxazolone challenge. Using real-time spinning disk confocal microscopy and two distinct fluorescent probes, we were able to simultaneously track two populations of rapidly moving cells, namely a dominant neutrophil population and a minor CD4 T cell population inside blood vessels during CS. We observed that both classical chemoattractants, C5a and LTB4, play a role in neutrophil adhesion during the early phase of the CS response. However, only C5a was responsible for the recruitment of a small population of CS-initiating effector CD4 T cells during the early phase. Blockade of C5a but not LTB4 activity also diminished the late phase CS response to oxazolone. Interestingly, adoptive transfer experiments revealed that C5a receptor expression on lymphocytes but not on tissue resident cells was essential for the development of a normal CS response. Thus, we report that C5a directly regulates effector CD4 T cell recruitment during the initial phase of the CS response.

We suggest that the recruitment of T lymphocytes within the very early phase of the contact hypersensitivity response is critical to creating a local microenvironment that is conducive to the later leukocyte recruitment and inflammation characteristic of the 24-h CS response. This is in line with previous observations that the transfer of CD4 T cells only within the first 2 h after CS initiation can restore the late phase inflammatory response in CD4-deficient mice (2, 3). Although the blockade of a number of chemokines such as CCL21, CCL17, MCP-1, IP-10, and MIP-1 ({alpha} and β) have been shown to diminish the late 24 h leukocyte recruitment during CS (5, 33, 34, 35), their specific role in the early 2-h CD4 T cell recruitment has never been explored due to the difficulty of tracking this population of leukocytes. A role for these chemokines in early recruitment, however, would seem unlikely as many of these chemokines are elevated slightly from baseline levels in a hapten-dependent manner at the 2-h time point but are present at much greater amounts 24 h after hapten challenge (5), consistent with a role in later effector cell recruitment. In line with this, Tsuji and colleagues (5) reported that early chemotactic activity from skin extracts is dependent on C5a, while later 24 h chemotactic activity is C5a independent. These authors postulated that C5a may be the key molecule that might recruit the initiating effector CD4+ T cells in vivo via mast cell and/or endothelial cell activation.

Using standard intravital microscopy, we identified a role for C5a, acting through the C5a receptor, in inducing the adhesion of an unspecified population of leukocytes during the early CS phase. Using the spinning disk confocal system, we could switch back and forth between two fluorescent channels to simultaneously examine the interactions of specific leukocyte subsets (neutrophils and CD4 T cells) with the endothelium in real time. On average in wild-type mice, for each rolling CD4 T lymphocyte ~10–18 rolling neutrophils were observed. This technique also revealed that the decreased adhesion observed in C5aR–/– mice was a result of a partial reduction in neutrophil adhesion and the complete reduction of effector CD4 T lymphocytes at 2 h after hapten challenge. The spinning disk technology was sufficiently sensitive to detect the abolishment of 90% of all leukocytes (all neutrophils) using an LTB4 receptor antagonist, whereas the few CD4 T cells remained adherent. Entirely consistent with the selective lymphocyte inhibition at 2 h was the reduction in tissue injury and cellular infiltration in the C5aR–/– mice, but not in LTB4-inhibited mice, 24 h after hapten challenge. This, in fact, reveals additional important information, namely that early neutrophil recruitment is not necessary for the progression of neutrophil or other leukocyte subset recruitment at the 24-h time point.

Interestingly, lack of C5a receptor blocked the recruitment of leukocytes into tissues but did not alter leukocyte adhesion within skin postcapillary venules or associated tissue swelling at the 24-h time point. Clearly, additional mechanisms are involved within the vasculature at this time point that are independent of the early T cell recruitment. Importantly, this suggests that the CD4 T cells initiate the prolonged 24-h leukocyte recruitment and tissue injury but do not mediate the inflammation seen in the vasculature and the subsequent vascular dysfunction (edema formation). This C5a receptor-independent vascular activity was also not due to LTB4 alone (Fig. 5, C and D) and could not be blocked when the LTB4 inhibitor was added to C5aR–/– mice (our unpublished data), ruling out redundancy. Interestingly, previous studies have revealed a dominant role for C5a only if suboptimal hapten dosing methods were used to induce CS (6, 11); however, these studies only examined edema as an endpoint. In the current study we have used a high concentration of hapten that is entirely consistent with a residual CS response seen in the C5aR–/– mice but restricted exclusively to the vasculature. Recently, studies have reported a second C5a receptor, C5L2, which can play both a proinflammatory and an anti-inflammatory role during immune responses (36, 37). Although C5a receptor deficiency resulted in the complete inhibition of CD4 T cell homing to the challenge site and subsequent tissue injury, it is possible that the lack of inhibition of leukocyte adhesion and edema formation at the 24-h point in C5aR–/– mice may reflect the role of the other C5a receptor, C5L2. Although the C5a receptor independent mechanism that mediates the late phase vascular inflammation remains unknown, based on other studies (8, 9, 10) it could also include the release of vasoactive mediators such as TNF-{alpha} and serotonin from mast cells and platelets. Indeed, it has recently been shown that mast cell activation can occur in a complement-independent manner during the CS response via B cell-generated, Ig-free light chains (38, 39, 40).

It has been previously postulated that C5a, likely through interaction with its receptors on mast cells and platelets, triggers the release of proinflammatory molecules that lead to the recruitment of effector T cells into the skin. The high immunizing dose of hapten used in this study does not generate an attenuated CS response in mast cell-deficient mice (our unpublished data). However, the CS response to a low immunizing dose is attenuated in mast cell-deficient mice (our unpublished data and Ref. 13) and could be dependent upon C5a stimulating mast cells as well as platelets. Interestingly, recent studies using this low immunizing dose have suggested that mast cells contribute to granulocyte recruitment rather than lymphocyte recruitment during the CS response (13). Indeed, in the current study we used adoptive transfer to show for the first time that the mechanism of C5a action is directly on effector CD4 T cells and not on tissue-resident cells. Given that both B and T lymphocytes are required to get a full CS response in naive mice, we transferred mononuclear cells derived from the spleens and lymph nodes of sensitized mice without isolating particular subsets. We have clearly shown that a CS response is initiated if sensitized donor cells derived from wild-type mice, but not C5aR–/– mice, are transferred to naive wild-type mice. Indeed, upon examination of the transferred mononuclear cell-endothelial cell interactions at the 2-h time point, C5aR–/–-derived cells clearly had an inability to adhere in the challenged skin microvasculature compared with wild-type cells. In these experiments all tissue-resident cells would be able to interact with generated C5a, suggesting that C5a interaction with mast cells or endothelial cells is not sufficient for the recruitment of effector CD4 T cells during the CS response. Conversely, a robust CS response was generated in naive wild-type and C5aR–/– mice that had been treated with sensitized wild-type cells, suggesting no need for the C5a receptor on tissue-resident cells.

In the current study, we have focused on the role of the C5a receptor during the initial stages of the effector phase of a CS response. However, the effects of C5a during the CS response are likely to be more widespread than just mediating leukocyte recruitment. Indeed, C5a may also play a role in the polarization of T cells. Recent studies have revealed that, in the absence of a C5a receptor, dendritic cells preferentially polarized T cells to a Th2 phenotype in a model of allergic asthma, suggesting that C5a may also play a role in the immunizing phase of an immune response (41, 42).

In this study, we use a relatively novel technology to track a very rare population of lymphocytes that cannot be detected by histology or standard intravital microscopy. Standard intravital microscopy is limited by its inability to accurately discriminate between the various leukocyte subpopulations. Multiphoton microscopy is extremely useful for studying distinct populations of leukocytes outside the vasculature, where dynamic events are sufficiently slow so that they can be observed using time-lapse systems, but it cannot be used to track leukocytes in the vasculature due to slow imaging speed. Recently, Frenette and colleagues (43) reported that high-speed, high-resolution multichannel fluorescence intravital videomicroscopy could be used to track different leukocyte subsets in response to a cytokine. We used similar spinning disk multichannel fluorescence intravital videomicroscopy (for the first time to our knowledge) to examine the very early dynamic events in blood vessels during CS. We report that this technique permits studying very rare populations of leukocytes simultaneously with more dominant populations during the initiation of an immune response. Moreover, we can determine selective inhibition of each cell type with various immunomodulators. In conclusion, using the multifluorescence system we report that while LTB4 blocks the recruitment of neutrophils at 2 h of CS, the main chemoattractant responsible for initiating CS-inducing CD4 T cell recruitment is C5a.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Canadian Institutes of Health Research and Canadian Institutes of Health Group. P.K. is an Alberta Heritage Foundation for Medical Research Scientist and a Canadian Research Chair recipient; M.U.N. is an Australian National Health and Medical Research Council CJ Martin Fellowship holder (284394) and is also an Alberta Heritage Foundation for Medical Research and Canadian Institutes of Health Research fellowship holder. Back

2 Address correspondence and reprint requests to Dr. Paul Kubes, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, T2N 4N1, Canada. E-mail address: pkubes{at}ucalgary.ca Back

3 Abbreviations used in this paper: CS, contact sensitivity; CP105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzl)-4-hydroxychroman-7-yl]-cyclopentane carboxylic acid; 5-LO, 5-lipoxygenase; LTB4, leukotriene B4. Back

4 The online version of this article contains supplemental material. Back

Received for publication August 8, 2007. Accepted for publication October 12, 2007.


   References
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 Disclosures
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