Memory peripheral Th2 immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. We have examined the localized immune response to a nematode parasite that encysts in the small intestine. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4+, TCR-αβ+ T cells surrounding the cyst. Laser capture microdissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host:parasite interface.
During an effective adaptive immune response it is thought that naive CD4 T cells differentiate to effector memory T cells that migrate to peripheral nonlymphoid tissues where the memory cells can rapidly respond to a subsequent challenge. Accumulation of CD8 T cells or IFN-γ-producing Th1 cells at peripheral sites of infection is well established (1). However, accumulation of polarized, IL-4-producing Th2 cells in peripheral nonlymphoid tissues during infectious disease is not well examined, partly because of the paucity of suitable models. Indeed, several reports have suggested that the Th2 response is more associated with B cell help in lymphoid tissue and that effector T cell migration to peripheral nonlymphoid sites of infection is more associated with inflammatory Th1 responses (2).
The primary response to the murine gastrointestinal nematode parasite, Heligmosomoides polygyrus (HP),3 is associated with chronic infection. However, if the parasite is cleared from the mouse with an antihelminthic drug, subsequent reinfection of the mouse with HP results in a memory response and worm expulsion by ∼2 wk after inoculation (3, 4). This clear distinction between primary and memory responses makes this infection a useful model for study of a functional memory Th2 response. Infection of mice with HP involves oral inoculation with third-stage larvae (L3) that migrate into the muscularis at the submucosal border of the jejunum of the small intestine and develop into adults over an 8-day period, after which the parasites migrate back to the gut lumen. An inflammatory cell infiltrate develops around the invading parasitic larvae, resulting in a border of cells that delineate a loosely defined, cyst-like structure (5, 6). Although the mechanism of worm expulsion remains uncertain, direct effects of IL-4 and IL-13 on gut tissue are probably important (7, 8, 9), and some studies have suggested that an effective memory response is also associated with delayed larval development before emergence into the lumen (5, 10). The CD4 T cell response has not been examined at peripheral sites of infection, but pronounced increases in CD4 T cell IL-4 and IL-13, but not IFN-γ, are detected in the mesenteric lymph node (MLN) as early as day 8 after primary inoculation and secondary challenge (11, 12).
Investigation of cytokine production at the localized site of parasite infection is difficult due to the short half-life and low expression level of IL-4 and IL-13 mRNA and protein. The relatively small number and size of cysts in the intestinal jejunum and the presence of other lymphoid tissue, including lamina propria and Peyer’s patch, make it difficult to extrapolate cytokine expression patterns in whole gut to changes that may be occurring at the host:parasite interface. The recent development of laser capture microdissection (LCM), which permits microscopic visualization and selection of specific tissue regions for mRNA analysis (13), provides a technology that makes it possible to assess Th2 cytokine gene expression in localized tissue microenvironments.
In the studies described in this report, we characterized a peripheral Th2 memory immune response. Our findings unexpectedly indicate that the Th2 response is initially associated with pronounced infiltration of neutrophils and macrophages to regions immediately adjacent to the parasite. In the memory, but not the primary, response, CD4+ T cells also accumulated as early as 4 days after HP inoculation, and LCM analysis showed pronounced CD4-dependent elevations in Th2 cytokines. These studies demonstrate that a highly polarized Th2 memory response can rapidly develop in peripheral nonlymphoid tissues during infectious disease.
Materials and Methods
Animals and treatments
Female BALB/c mice (Small Animal Division, National Cancer Institute, Fredrick, MD) were used for each experiment and were inoculated orally with 200 L3 as previously described (14). Separate groups of HP1°-infected mice were treated with the antihelminthic drug, pyrantel pamoate, at 14 days after inoculation (Rx), and were reinfected orally with 200 L3 at 50 days after inoculation (HP2°) or were not infected (HP1°+Rx). The gut tissue was collected on day 4 after secondary inoculation. Some groups of HP-challenged mice were treated with anti-CD4 Ab (GK1.5) on day 3 and examined on day 4 after secondary inoculation.
Preparation of frozen tissue blocks and sectioning
The gut tissues were taken from the jejunum of HP-infected mice, slit longitudinally, prepared using the Swiss-roll technique (15, 16), embedded in Tissue-Tek OCT compound (Sakura Finetek U.S.A., Torrance, CA), frozen on dry ice-acetone, and stored at −80°C. For LCM, 4-μm tissue sections were cut from frozen blocks using an HM505E cryostat (Richard-Allan Scientific, Kalamazoo, MI).
Four-micron frozen tissue sections of Swiss-rolled jejunum of the small intestine were fixed in cold acetone and then stained with anti-CD11c-PE, anti-Gr-1 PE, anti-Gr-1-FITC, anti-B220 (6B2)-FITC, anti-TCR-γδ-PE, anti-CD8-PE, anti-TCR-β-PE, and anti-CD4 PE (BD PharMingen, San Diego, CA) or F4/80-PE (5 μg/ml; Caltag, Burlingame, CA), followed by 10 μg/ml of 4′,6-diamido-2-phenylindole hydrochloride (DAPI; Roche, Indianapolis, IN) for fluorescence analysis as previously described (14). For composite photos, a 4-μm section of intestinal jejunum prepared as a Swiss roll was mapped using scanning software designed by J. Czege (Biomedical Instrumentation Center, Unformed Services University of the Health Sciences). As previously described for lymph node tissue (17), mapped regions of the intestinal tissue section were then individually photographed at ×200 magnification using a SPOT2-cooled CCD camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a Leica DMRXA (Leica Microsystems, Bannockburn, IL) computerized fluorescence microscope and using SPOTAdvance software (Diagnostic Instruments). Each fluorescent channel was photographed separately, and the three sets of ×200 images were merged using TIFFany3 software (Caffeine Software, Santa Clara, CA) to create the final composite photo of intestinal tissue.
LCM and real-time RT-PCR
Cryosectioned tissue sections of a Swiss roll preparation of the jejunum of the small intestine were dehydrated and stained for H&E (Sigma-Aldrich, St. Louis, MO). LCM of stained sections was performed on a PixCell II (Arcturus Engineering, Mountain View, CA), and captured cells were transferred to CapSureTM LCM Caps (Arcturus Engineering, Mountain View, CA). The LCM cap was inserted into a 0.5-ml microcentrifuge tube containing RNA isolation solution, and total RNA was extracted using an RNA isolation kit (Stratagene Cloning Systems, La Jolla, CA). In other experiments whole MLN tissue was homogenized in RNAzol B (Cinn/Biotecs, Friendswood, TX) and purified according to the manufacturer’s instructions. Total RNA was then reverse transcribed as previously described (18). Real-time PCR kits (PE Applied Biosystems, Foster City, CA), specific for individual cytokines or rRNA, were used to quantitate differences in gene expression; all data were normalized to constitutive rRNA values and a PE Applied Biosystems 7700 sequence detector was used for target mRNA amplification.
A specific immune cell infiltrate architecture develops at the host parasite interface of HP-challenged mice
The HP L3 encyst within the muscularis that borders the submucosal region of the jejunum of the small intestine. Previous studies have reported that an inflammatory infiltrate develops around the worm, which includes both macrophages and polymorphs, and that resistance after secondary challenge may be associated with delayed maturation of parasitic larvae within the intestinal mucosa (4, 5, 10). To further characterize the cells infiltrating the cyst, we used immunofluorescent staining with specific Abs detecting cell surface markers on cells associated with the immune response. Fig. 1,A shows a section of jejunum from a Swiss roll preparation on day 4 after HP challenge inoculation that was stained for CD4 (red), Gr-1 (green), and DAPI (blue) as a background stain. The high resolution microphotograph shown is actually a composite of multiple photos (×200) taken individually and tiled together as described in Materials and Methods. Areas of cysts containing individual larva are readily observed as Gr-1+ cells specifically infiltrate them. Dark regions inside the cyst are sections of the actual parasite. CD4+ T cells are greatly increased in regions of the lamina propria near the cyst, indicating specific localization of CD4+ T cells to the area surrounding the invading parasite and associated granulocytes during challenge inoculation. High power analysis (×1000) of H&E-stained, paraffin-embedded sections showed predominantly neutrophils and macrophages, with occasional, but scarce (<1%), eosinophils (Fig. 1,B). Surprisingly, although eosinophils were rare in the region around the cyst, they were abundant in the lamina propria (Fig. 1 C). As expected, eosinophils in the lamina propria did not stain for Gr-1.
Additional phenotyping was performed and is shown in Fig. 2. Specific staining for macrophages (F4/80) showed that cells of this phenotype were localized to regions of the lamina propria immediately surrounding the cyst and that they also infiltrated the cyst region (Fig. 2,B). Gr-1+ cells also infiltrated the cyst, but did not dual stain with F4/80, indicating that these two Ags were expressed on distinct populations (Fig. 2,B, inset). Gr-1+ cells also stained CD11b+ (Fig. 2,E). The inset of Fig. 2,E shows considerable yellow fluorescence resulting from both cell surface Ags being expressed on the same cell. The observation that Gr-1+ cells were CD11b+ and F4/80− is consistent with a neutrophil phenotype. Both the fluorescent and the H&E staining indicated that there was pronounced infiltration by macrophages and neutrophils in approximately equal numbers. CD11c+ dendritic cells were restricted to the lamina propria area outside the cyst region, showing a similar distribution to CD4+ cells (Fig. 2,C). TCR-γδ cells (Fig. 2,F), B cells (data not shown), CD8 T cells (data not shown), and NK cells (data not shown) were not detected. TCR-αβ+ T cells (Fig. 2,D) showed the same distribution as CD4+ T cells, and two-color staining confirmed that the CD4+ cells were predominantly TCR-αβ+ (data not shown). Jejunum samples were also examined on day 4 after primary HP inoculation. Similar, but markedly reduced, populations of infiltrating cells were observed (data not shown), and CD4 T cells had not accumulated to a significant degree around the cyst (Fig. 2 G). Thus, an acute inflammatory response associated with high numbers of CD4+ T cells and a highly specific immune cell architecture develops as early as day 4 after HP challenge inoculation.
IL-4 and IL-13 gene expression in different microenvironments of the small intestine after HP priming and challenge
Resistance to HP, which occurs after secondary challenge, but not after primary inoculation, is immune mediated and CD4+ T cell dependent (19). The early activation and maintenance of this robust type 2 immune response, which includes increased expression of IL-4, but not IFN-γ, in the draining lymph nodes on day 10 after challenge inoculation are required for controlling the level of infection (4, 7, 12, 20).
To identify mucosal microenvironments outside the secondary lymphoid tissues, where Th2 cytokines were expressed, we used LCM. This technique allows microscopic collection of individual tissue samples ∼7.5 μm in diameter, which usually includes one to three cells (see Fig. 3). To examine the gene expression of individual microenvironments in the small intestine of HP-infected mice, cells were captured from 1) lamina propria; 2) Peyer’s patch; 3) the area immediately surrounding the cyst, including CD4+, TCR-αβ+ cells in HP-challenged mice; and 4) the area inside the cyst, containing primarily macrophages and neutrophils, but no T cells. Multiple samples (∼1000) were taken in the selected regions for each mouse and pooled for RNA isolation.
RNA was purified from pooled LCM samples and reverse transcribed, and IL-4, IL-13, IFN-γ, and rRNA were assessed by real-time RT-PCR. All data were normalized to constitutive rRNA values. In each experiment, all samples were measured in the same assay for each cytokine, allowing direct comparison between different groups or tissues. In all groups and tissues examined, no elevations were detected for IFN-γ (data not shown). On day 4 after primary HP-inoculation, IL-4 and IL-13 were detected in the Peyer’s patch, but not in the lamina propria, inside the cyst, or in the area immediately surrounding the cyst (Fig. 4). Similarly, although residual cysts were present in the intestinal muscularis of the control group on day 54 after primary inoculation (did not receive a challenge inoculation), no elevations in IL-4 and IL-13 were detected in Peyer’s patch, lamina propria, or inside or surrounding the residual cysts (data not shown). However, on day 4 after HP secondary challenge, pronounced IL-4 and IL-13 elevations occurred in Peyer’s patch and lamina propria and inside the cyst. Even higher elevations in these Th2 cytokines were detected in the nonlymphoid tissue immediately surrounding the cyst. Thus, at this early time point of the memory response, but not the primary response, Th2 cytokine gene expression was pronounced at the localized site of the host:parasite interface. Using the LCM technique, we have also examined cytokine gene expression at a later time point after secondary challenge (day 12). Our results showed that IL-4 and IL-13 mRNA elevations were substantially reduced in the area surrounding and inside the cyst, with no elevations in IFN-γ detected (data not shown). This is most likely a consequence of the immune stimulus, namely the worm, leaving the cyst region to occupy the intestinal lumen on day 8 after challenge inoculation.
Anti-CD4 treatment inhibits Th2 cytokine elevations
To examine the contribution of CD4+ T cells to the increased Th2 cytokine gene expression in tertiary tissue sites after secondary challenge with HP, HP-challenged mice were administered anti-CD4 Abs at doses (1 mg) that have previously been shown to effectively inhibit the host protective memory response and block elevations in MLN Th2 cytokine gene expression (19). As shown in Fig. 5, mice that received anti-CD4 Abs on day 3 after secondary HP inoculation and were examined on day 4 showed markedly reduced IL-4 and IL-13 elevations (>50-fold) in the region surrounding the cyst, where the CD4+, TCR-αβ+ T cells were localized. It should be noted that although gene expression was reduced after CD4 T cell depletion, residual elevations were still detectable, suggesting that blockade was not complete or that non-CD4 cells may contribute slightly to elevations in cytokine gene expression. After anti-CD4 Ab treatment, marked decreases were also observed inside the cyst, suggesting that the presence of CD4+ T cells surrounding the cysts was required to induce increased cytokine gene expression by cell populations inside the cyst. It should also be noted that granulocyte infiltration was not reduced or changed after anti-CD4 Ab administration during the HP challenge response (Fig. 2,H). This is similar to our findings for the HP primary response, where neutrophils infiltrated the cyst on day 4 after HP primary inoculation, although CD4+ T cell expansion was not observed (Fig. 2 G).
Th2 cytokines are up-regulated as early as day 2 in the MLN during the HP memory response
To examine whether a Th2 memory response in the draining lymph node precedes the Th2 memory response observed in the cyst on day 4 after HP inoculation, MLNs were collected from HP-primed and -challenged mice at several time points after inoculation. As shown in Fig. 6, IL-4 and IL-13 gene expression were elevated as early as day 2 after HP secondary challenge, whereas greatly reduced increases were observed after primary immunization. These early Th2 cytokine elevations in the MLN, specifically associated with the memory response, suggests that memory T cells initially stimulated in the draining lymph nodes may provide a source for Th2 memory cells at the host:parasite interface.
The peripheral memory CD4 T cell response is one of the most important outcomes of adaptive immunity during infectious disease, providing the basis for rapid natural immune protection and vaccine development. We have characterized the cell populations at the host:parasite interface during a peripheral memory Th2 immune response to an intestinal nematode parasite and used LCM to characterize localized cytokine gene expression at specific sites in peripheral mucosal tissues from infected mice. Our findings demonstrate that 1) during the Th2 memory response a specific microenvironment rapidly develops with neutrophils and macrophages infiltrating and CD4+, TCR-αβ+ T cells surrounding the cyst-like structure associated with the invading parasite; 2) after challenge, the CD4 T cells at the host:parasite interface are highly polarized, expressing pronounced elevations in Th2 cytokines; and 3) the rapid CD4 T cell response and associated cytokine elevations are not observed during the primary response, although neutrophils and macrophages still accumulate at the host:parasite interface.
Previous studies have suggested that effector memory T cells develop in lymph nodes during the primary response and migrate to peripheral tissues, where they can rapidly respond to secondary stimulation. This model is based on responses that stimulate CD8 memory T cells (1, 21) or, more recently, CD4 Th1 memory cells (22, 23); studies of the latter population frequently rely on potentially nonphysiological approaches involving responses of Ag-specific transgenic T cells that have been transferred to Ag-immunized recipients. In the results reported in this paper we have defined an experimental system for examining peripheral memory Th2 effector cell function at the host:parasite interface. The response to this natural murine parasite has previously been shown to be highly polarized, with elevations in IL-4, but not IFN-γ or IL-10, in the draining lymph nodes after either primary or challenge HP inoculation (11, 12). We have used this model system to describe for the first time the development of a highly polarized peripheral Th2 memory response during infectious disease. The rapid development of this response after challenge, but not primary, inoculation suggests that Th2 memory cells have the capability to rapidly migrate to peripheral nonlymphoid sites and differentiate into potent sources of Th2 cytokines. This scenario is analogous to models for peripheral memory Th1 responses, suggesting that Th2, as well as Th1, memory cells are capable of rapid migration and effector cell function at sites of inflammation. As elevations in Th2 cytokines were detected in the MLN as early as day 2 after challenge inoculation, it remains uncertain whether the CD4 cells at the site of infection migrated from the lymph nodes or from peripheral nonlymphoid tissues. The observation that IL-4 and IL-13 are elevated, whereas IFN-γ is not, in both lymphoid and peripheral nonlymphoid tissues after challenge inoculation suggests that in the Th2 response to HP, central memory cells as well as peripheral memory cells exhibit a highly polarized Th2 response. This is in contrast to studies in some other model systems, where central memory cells in the lymph nodes may initially produce a mixed Th1/Th2 cytokine pattern after challenge immunization (24, 25).
In the memory response, pronounced increases in IL-4 and IL-13, but not IFN-γ, were observed in lamina propria, Peyer’s patch, and the microenvironment of the invading parasite, indicating that this particular pathogen triggers a highly polarized Th2 cytokine pattern that rapidly develops in different mucosal secondary lymphoid and tertiary tissue sites. Interestingly, although the developing larval parasite resides in nonlymphoid tissue, infiltrating cells at the host:parasite interface raised Th2 cytokine mRNA to levels at or above those obtained in mucosal lymphoid tissues. The degree of Th1/Th2 skewing appears to vary greatly depending on the particular invading pathogen. For example, the peripheral response to influenza is polarized toward a Th1 pattern (26), whereas the response to Listeria is associated with elevations in both Th1 and Th2 cytokines (27). However, a caveat is that cytokine expression in these reports was examined using in vitro restimulation assays, which may result in the production of cytokines not expressed in vivo.
The use of LCM permitted direct assessment of cytokine mRNA levels using highly sensitive fluorogenic RT-PCR analysis at the specific site where the parasite encysted in the small intestine. Less sensitive techniques, such as in situ hybridization or protein staining, are usually not practical with Th2 cytokines due to their short mRNA and protein half-life. Conventional analysis of gene expression using more sensitive techniques are limited to much larger regions of tissue (28), making it difficult to distinguish specific microenvironments, in this case regions where host:parasite interactions actually occur in the intestinal tissue. Other approaches for the detection of IL-4 expression have used transgenic reporter mice or knockin mice expressing green fluorescent protein linked to IL-4 (29). However, in these studies the reporter protein may not be secreted similarly to the cytokine, or its half-life may be different. LCM has the distinct advantage, in that one can actually examine individual regions of interest from undisturbed specific tissue microenvironments in normal mice and directly assess in situ cytokine gene expression using highly sensitive, PCR-based techniques.
A characteristic and consistent pattern of specific immune cell infiltrates was observed at the host:parasite interface during the memory Th2 response. Although macrophages both infiltrated and surrounded the cyst region, neutrophils were predominantly observed only in the region immediately around the parasite. Of note, eosinophils were restricted to the lamina propria and were rare to absent at the host:parasite interface on day 4 after inoculation. Macrophages and neutrophils are generally associated with Th1 inflammatory responses and down-regulated production of Th2 cell cytokines (30, 31, 32, 33). However, recent studies have suggested that IL-4 and IL-13 can induce alternatively activated macrophages that show distinct phenotypes from macrophages activated by IFN-γ (34, 35, 36). Recent findings have also indicated that activated neutrophils can produce IL-4 (37) and that they accumulate during chronic allergic responses (38). Our findings indicate that neutrophils and macrophages are major infiltrating populations at early stages of the peripheral Th2 as well as the peripheral Th1 inflammatory response, demonstrating the plasticity of these responding populations during infection. Interestingly, a previous study has suggested that neutrophils may be important in protective immunity against HP (39). Macrophage infiltration is also observed in the granulomatous response to schistosome eggs; however, at early stages of this primary response a mixed Th1/Th2 cytokine pattern is observed (40, 41), whereas the memory response to HP is not associated with elevations in Th1 cytokines even at early stages of the response. It should be noted that the HP infectious model system is particularly useful for CD4 Th2 cell memory studies, as the parasite can be completely cleared from the gut with anthelminthic drugs before subsequent challenge inoculation. It is of interest that a number of cell populations, often associated with Th1 mucosal immune responses, including NK T cells and TCR-γδ cells (42, 43, 44), were not detected at the host:parasite interface, suggesting that in this highly polarized Th2 memory response these cells do not play a major role at the site of infection. In terms of adaptive immunity, it appears that the major infiltrating population is CD4+, TCR-αβ+ T cells. This distinct response should provide a useful model for examining immune cell interactions at the host:parasite interface during the acute memory Th2 inflammatory immune response.
The studies described in this report demonstrate that a polarized Th2 peripheral memory response associated with a distinct immune cell architecture rapidly develops in the localized microenvironment of an invading parasite, and they establish the use of LCM as a technique for examining peripheral memory Th2 cell distribution and function in situ.
This work was supported by National Institutes of Health Grant AI31678. The opinions or assertions contained within are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense or the Department of Agriculture.
Abbreviations used in this paper: HP, Heligmosomoides polygyrus; DAPI, 4′,6-diamido-2-phenylindole hydrochloride; L3, third-stage larvae; LCM, laser capture microdissection; MLN, mesenteric lymph node; Rx, inoculation.