Introduction

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The successful expression of acquired protective immunity to tuberculosis infection involves the intimate interaction between infected macrophages and effector (protective) T cells. It is clear from many studies that T cells, and more specifically CD4 T cells, mediate the bulk of the protective and anamnestic host responses (1). These cells clonally expand in the presence of interleukin (IL)-2 and respond to the macrophage cytokine IL-12 by producing large quantities of inteferon (IFN)- (7). In humans, a reduction in CD4 T cells or an inability to mediate signals via the IL-12/IFN- pathway renders the individual highly susceptible to mycobacterial infection (10).

There is, however, evidence to support a role for other T cells in pulmonary infection. CD8 T cells harvested from immune mice transferred prolonged survival in an acute aerosol infection model (3), and naive CD8 T cells protected athymic mice against intravenous infection (11). In addition, work by Flynn and colleagues (12) clearly demonstrated a substantially increased susceptibility of 2-microglobulin gene-disrupted (2-m-KO) mice to intravenous infection with virulent Mycobacterium tuberculosis. These mice had increased growth of the bacilli within lung tissue and exhibited substantial necrosis and tissue damage in that organ.

Owing to the inability of the 2-m-KO mice to sensitize CD8 T cells, it was reasonable to conclude at that time that the increased susceptibility was due to the lack of this subset of T cells. To confirm this, we compared 2-m-KO and CD8 gene-disrupted (KO) mice in a relevant low-dose infection model. Surprisingly, our data failed to confirm this hypothesis. Although CD8-KO mice allow increased bacterial growth in the lung late in infection, the loss of resistance in the 2-m-KO mice occurs much earlier and is more severe. This loss of resistance in the 2-m-KO mice appears to be associated with a failure to adequately recruit lymphocytes, but not macrophages, to sites of bacterial infection in the lungs.

As a result of these findings, we hypothesized that other molecules associated with 2-microglobulin may play a role in moderating the development of pulmonary granulomas. To test this hypothesis, we infected CD1 gene-disrupted mice in the same manner as the 2-m-KO and CD8-KO mice. These mice lack the 2-microglobulin-associated nonpolymorphic major histocompatibility complex (MHC) class 1-like molecule that serves to sensitize natural killer (NK) 1.1-positive, T cells (13). These NK T cells have been shown to release several cytokines and chemokines very rapidly upon activation and have been suggested to play a role in initiating and/or modulating the acquired immune response (14). We show here, however, that these mice exhibited no increased susceptibility to M. tuberculosis infection nor did they show any evidence of altered granuloma formation. We therefore hypothesize that a previously unrecognized, non-CD8, non-CD1, 2-microglobulin-dependent mechanism is operative early in infected lungs and serves to control the efficient accumulation of lymphocytes into this tissue.

	Materials and Methods

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Mice and Infections

2-m-KO mice on both the B6/129 background and the C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, ME). Littermate control mice, C57BL/6 mice, and founder CD8-KO mice were also purchased from Jackson Laboratories. CD8-KO mice were bred in the Painter Center animal facility at Colorado State University (Fort Collins, CO) and were consistently negative for CD8-positive cells by flow cytometry. CD1-KO mice were generated and tested as described (13), bred at Princeton University animal facility (Princeton, NJ) and transferred to Colorado State for infection. The CD1 gene disruption was backcrossed onto the C57BL/6 background six times. Mice were housed in the BL3 facility and given mouse chow and water ad libitum.

A virulent strain of M. tuberculosis (Erdman) was grown from a low-passage-number seed lot in Proskauer-Beck liquid medium to mid-log phase, aliquoted, and frozen at 70°C. Mice were infected using a Glas-Col aerosol generator (Glas-Col, Terre Haute, IN) such that 100 bacteria were deposited in the lungs of each animal (8). The numbers of viable bacteria in target organs were determined at various time points by plating serial dilutions of partial organ homogenates on nutrient Middlebrook 7H11 agar and counting colonies after 20 d of incubation at 37° C. A Day 1 count was performed to determine the infecting dose.

Some mice were treated with a blocking anti-CD1 antibody (rat immunoglobulin [Ig] G1 clone 20H2, 1 mg/mouse intraperitoneally) (15) at Days 1, +1, 3, and 5 of an aerosol infection. Control mice received rat IgG1 isotype control antibody following the same regimen.

Histologic Analysis

The lower right lobe of each mouse was inflated with 10% neutral-buffered saline and processed routinely for light microscopy. Sections were then stained with hematoxylin and eosin. Slides were examined without knowledge of experimental group and subjectively graded for both quantity and quality of cellular accumulation. Repeat evaluations were performed to confirm that grading was reproducible.

In addition, sections from formalin-fixed tissue were deparaffinated and placed in 10 mM sodium citrate buffer (pH 6), followed by pressure cooking for exactly 1 min. After blocking for 20 min in 1% H₂O₂ solution, slides were incubated with appropriately diluted polyclonal rabbit antimouse nitric oxide synthase (NOS)2 (Genzyme-Virotech, Russelsheim, Germany) in Tris-buffered saline/10% fetal calf serum for 30 min in a humid chamber. As bridging antibody, appropriately diluted goat-antirabbit-IgG-peroxidase (Dianova, Hamburg, Germany) and as tertiary antibody, diluted rabbit-antigoat-IgG-peroxidase (Dianova) was used in sequential incubations of 30 min each. Development was performed with diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and urea superoxide (Sigma), and hemalum was used to counterstain the slides. Isotype-matched antibody was used on some sections to confirm that staining was specific (data not shown).

Isolation of Messenger RNA and Detection of Cytokine-Specific Message by Reverse Transcriptase/Polymerase Chain Reaction

Infected and control tissues were excised, placed in Ultraspec (Cinna/Biotecx, Friendswood, TX), homogenized, and RNA was extracted as described previously (7). One microgram of total RNA was reverse-transcribed, diluted and underwent polymerase chain reaction (PCR) expansion of cytokine-specific complementary DNA (cDNA). The amount of cytokine-related product was determined by the exposure of blotted cDNA PCR product to fluorescein-tagged cytokine sequence-specific probe. The enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) was used to detect the presence of fluorescein by horseradish peroxidase-conjugated antibody. The resultant light signal was detected using Hyperfilm (Amersham). The number of cycles that generate a log-linear relationship between the signal on film and the dilution of the sample was determined empirically (7). Data are expressed as the "fold increase" in signal for experimental points relative to the control value from uninfected lung tissue. The significance of the fold increase over uninfected tissue was determined by an unpaired Student's t test comparing the means of the signals from uninfected versus infected tissue.

	Results

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Increased Bacterial Growth in 2-Microglobulin Null Mice after Infection via the Aerogenic Route

Figure 1 represents data from two independent experiments. In each case, gene-disrupted and control animals were exposed to an aerosol infection that resulted in the deposition of about 100 bacteria into the lungs. Both C57BL/6 and B6/129 mouse data were pooled, as they did not differ from each other. CD8-KO mice were not significantly more susceptible to the aerogenic infection up to Day 55 but did show increased bacterial growth at later time points starting from Day 75 (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 1.   Mice lacking 2-microglobulin are more susceptible to aerogenic infection than are mice lacking CD8. Mice were exposed to an aerogenic infection with virulent M. tuberculosis such that each mouse received roughly 100 bacteria in the lung. Lungs were harvested from the mice at several time points and plated to determine the number of colony-forming units per lung. Data from 16 control C57BL/6 and B6/129 mice were pooled (solid bars), as there was no significant difference between the mean values of these two groups (Student's t test, P > 0.05). The mean values for eight mice per time point are shown for both the CD8-KO (striped bars) and the 2-m-KO (gray bars) mice. A statistically significant difference was observed between the 2-m-KO mice and the control and CD8-KO mice (asterisks) (Student's t test, P < 0.05).

In the 2-m-KO mice, the bacterial numbers increased more rapidly than in either the control mice or the CD8-KO mice. The difference was significant by Day 30 (P < 0.05). After that time, although the infection was apparently contained and did not result in the death of any animals, bacterial loads remained elevated through Day 90.

Because these data implied that the early loss of resistance in 2-m-KO mice was not due to a lack of the CD8 molecule, we then assessed the effect of disrupting only the CD1 molecule on murine resistance to an aerogenic mycobacterial infection. Figure 2 shows the pooled data from two independent experiments using C57BL/6 mice, heterozygote (CD1+/) mice, and homozygote CD1/ mice. There was no difference in bacterial growth either very early (Day 7) or during the control phase of infection (Day 23 or Day 44) in the lungs or spleens of the CD1-KO mice. In addition, mice treated with a blocking antibody to CD1 did not exhibit any increased susceptibility during the early stages of infection (Table 1).

View larger version (40K): [in this window] [in a new window]   Figure 2.   Mice lacking CD1d1 are not more sensitive to aerogenic infection than are control mice. Mice were infected and lungs harvested as in Figure 1. There was no statistically significant difference between the mean colony-forming unit values for control C57BL/6 mice (solid bars), heterozygote mice (striped bars), or the CD1-KO mice (gray bars) (Student's t test, P > 0.05) over the course of the experiment. The data shown are a representative result from two separate experiments.

Histologic Analysis of Lung Tissues after Aerosol Infection

The histologic appearance of lung tissues was followed over the course of infection. In control mice, mild interstitial pneumonia was observed at 15 d, which was followed after 20 to 30 d by the gradual emergence of the granulomatous response. This response was characterized by the presence of organized rafts of lymphocytes within fields of epithelioid macrophages (Figures 3A and 3B). The response was similar in the CD8-KO mice, with the exception that as the infection progressed more lymphocytes were seen within the granuloma, and large perivascular lymphocytic cuffs were seen around adjacent blood vessels (Figure 3C). In addition, CD1-KO mice developed a granulomatous response in the lungs in a similar manner to control mice (data not shown).

View larger version (153K): [in this window] [in a new window]   Figure 3.   Few lymphocytes are observed in the early granulomas of the 2-m-KO mice. Representative fields from the lungs of C57BL/6 (A), B6/129 (B), CD8-KO (C), and 2-m-KO mice (D) 55 d after infection. Mice were infected as in Figure 1, and samples for histology were taken at each time point. Note the florid lymphocyte accumulations in A, B, and C (arrowheads) and the predominance of macrophages and absence of lymphocytes in plate D. Hematoxylin and eosin staining was used.

Examination of 2-m-KO mice early in infection revealed a similar development of mild interstitial pneumonia, followed gradually by the accumulation of large numbers of epithelioid macrophages. Few lymphocytes were observed, predominantly in the perivascular region (Figure 3D). As the infection progressed, the perivascular cuffing grew more prominent but at no time was there any evidence of lymphocyte influx beyond the spaces adjacent to the blood vessels and into the epithelioid macrophage field (Figures 4A, 4B, and 4C). Furthermore, from about Day 30 onwards, these macrophage fields contained substantially increased numbers of degenerative "foamy" macro-phages (Figure 4C) with concordant necrosis. Probably secondary to this necrosis, more neutrophils were observed in these sections. Interestingly, the ability of the epithelioid macrophages to express the inducible NOS (iNOS) gene product was unimpaired in the granulomas of 2-m-KO mice, as determined by immunohistochemistry (Figure 5).

View larger version (99K): [in this window] [in a new window]   Figure 4.   As disease progresses, perivascular cuffing of lymphocytes becomes prominent in the lungs of the 2-m-KO mice. Lung sections from 2-m-KO mice at 30 (A), 55 (B), and 90 d (C) after aerosol infection. The paucity of lymphocytes within the granuloma is clear, as is increasingly evident cuffing of lymphocytes (black arrowheads) around the blood vessels (white arrows). Hematoxylin and eosin staining.

View larger version (148K): [in this window] [in a new window]   Figure 5.   Both control and 2-m-KO mice express iNOS within the lung granulomas. Formalin-fixed tissue samples were processed for immunohistochemical analysis as described. The images represent lung sections from 2-m-KO (A) and control mice (B) at Day 55 of infection. The dark brown stain indicates the presence of immunoreactive iNOS protein. Expression of this enzyme is focused in the macrophage accumulations in both groups of mice.

Expression of Both Messenger RNA for IFN- in Infected Lungs and Secretion of IFN- by Antigen-Specific Cells

To determine whether the increased susceptibility of the 2-m-KO mice was a result of ineffective induction of IFN-, the levels of cytokine-specific messenger RNA (mRNA) in the lungs of each mouse were determined. In two separate experiments, the kinetics and magnitude of IFN- mRNA expression were not statistically different between the control mice and either the 2-m-KO mice or the CD8-KO mice. The relative fold increase in signal for both Day 30 and Day 55 infected lungs is shown in Table 2.

Changes in mRNA levels for the chemokine monocyte chemotactic protein (MCP)-1 was also measured and a moderate, but significant, early increase in mRNA signal for this macrophage chemokine (Figure 6) was detected in the lungs of the 2-m-KO mice as compared with either the control or CD8-KO mice.

View larger version (20K): [in this window] [in a new window]   Figure 6.   2-m-KO mice exhibit earlier and enhanced expression of mRNA for the chemokine MCP-1 in response to infection. Lung samples were taken from each of the control mice (closed circles), 2-m-KO mice (open triangles), and CD8-KO mice (closed squares), and RNA was extracted. The mean fold increase over Day 0 pixel values for MCP-1 is shown at three time points (n = 4). The statistical significance of the increase in mean experimental pixel values over the mean Day 0 pixel values for both MCP-1 was determined by Student's t test (*P < 0.05). The statistical significance of the differences between the mean fold increase in control and gene-disrupted mice at each time point was also determined using the Student's t test (P < 0.05). Equivalent amounts of cDNA from each mouse were compared as determined by the signal obtained for the housekeeping gene hypoxanthine guanine phosphoribosyl transferase.

	Discussion

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The results of this study show that 2-m-KO mice are more susceptible than control mice to a low-dose aerosol infection with M. tuberculosis. In addition, the data provided evidence that the underlying mechanism resulting in this susceptibility was related not to an inability to generate protective immunity per se but seemed to be associated instead with an apparent inability to focus lymphocytes within infected lesions in the lung. Subsequent experiments revealed that this deficiency was not related to loss of CD8 or CD1 molecules, despite the association of 2-microglobulin with these molecules.

The evidence supporting this hypothesis was the striking difference in the histologic appearance of the lungs harvested during the early phase of infection. The CD8-KO, CD1-KO, and control mice all exhibited the expected granulomatous response (16) with the only difference being a possible increase in lymphocyte infiltration seen in CD8-KO mice. In stark contrast, however, the 2-m-KO mouse lungs showed a progressive macrophage influx in the virtual absence of any lymphocytes. As this response proceeded, lymphocytes did accumulate but they remained in gradually expanding perivascular cuffs and failed to infiltrate the macrophage-dominated lesion. Within these lesions, many of the macrophages had a histiocytic or vacuolated appearance, some had become multinucleated giant cells, and many appeared to be degenerative.

Intriguingly, despite the reduced ability of the 2-m-KO mice to control the early growth of the infection, IFN- mRNA was expressed in the lung with the same kinetics as seen in the control mice. In addition, this IFN- was able to mediate expression of iNOS in the epithelioid macrophages of both the control and 2-m-KO pulmonary granulomas. To explain this, it is reasonable to speculate that the lymphocytes accumulating close to the arterioles adjacent to the macrophage granulomas were the source of this cytokine.

Several lines of evidence, including the data reported here, allow us to develop the hypothesis that there are two phases of the immune response in the lungs after tuberculosis infection. A very early, presumably innate response appears to require 2-microglobulin but is independent of the CD8 molecule. In its absence, the bacterial load in the lungs rapidly increases, and the histologic appearance of the lungs is dramatically altered. Because our data seemed to exclude CD8, we then pursued the idea that the consequences of 2-microglobulin disruption were operative via the CD1 molecule. This molecule requires 2-microglobulin to be expressed (17), is able to bind unusual hydrophobic motifs (18), and is able to stimulate NK T cells (19). This natural T-cell subset occurs in both mice and humans, and responds rapidly to stimulation by the release of cytokines; as a result, it is thought to represent a bridge between the innate response and the acquired response (14). That our hypothesis was incorrect, however, was shown by experiments in which mice lacking the CD1d1 molecule (the only CD1 molecule expressed by C57BL/6 mice [20]) were able to control early infection in a manner similar to that of control mice. Moreover, the histologic appearance of the lungs during the infection did not differ from those of the control animals.

Our data indicate that presentation of mycobacterial antigens by CD1 molecules to NK T cells does not appear to play a role in early immune responses to tuberculosis in the murine lung. This is consistent with a recent report by Behar and colleagues (21), which showed that even when given a large intravenous dose of M. tuberculosis, CD1-KO mice were not more susceptible than control mice. There are, however, several other mechanisms that may be involved in the very early innate response during pulmonary infection. For example, there are several other nonclassic MHC class 1-related molecules that require 2-microglobulin for expression and that are associated with recognition of bacterial products or altered self (22). Whereas the evidence presented here indicates that CD8-dependent cells are not involved in the early resistance mechanism, there are other cell populations, such as T cells, that could potentially recognize nonpolymorphic class 1-like molecules.

While there is increasing evidence that nonpolymorphic, 2-microglobulin-dependent MHC-like molecules may provide an early warning of bacterial invasion or tissue damage (22), it remains completely unknown how this is achieved. Our working hypothesis is that bacterial antigens (probably easily detachable lipoglycans or lipoproteins from the outer surface of the cell envelope) are presented to an as yet undefined (presumably) T-cell subset. This presentation then leads to the development of local changes that result in the influx of mononuclear cells (23). This is probably a complex event, involving increased permeability of the local capillary bed, expression of adhesion/integrin molecules, and the production of chemokines that will preferentially attract lymphocytes and monocytes rather than neutrophils (24).

In the current study, we observed an increased expression of the chemokine MCP-1 in the 2-m-KO mice, in association with the increased bacterial load and florid macrophage influx. That this macrophage influx is not matched by an equally strong lymphocyte accumulation suggests that there may be a deficiency in the more T cell- directed chemoattractants. This might help to explain the capacity of accumulating lymphocytes to adhere to and cross the local arteriolar vessels but then fail to move through the intracellular matrix and into the developing granulomas. This inability of lymphocytes to move into the granuloma would also explain the results of Flynn and colleagues (25) who found that the 2-m-KO mice were not protected in the lungs by Calmette-Guerin bacillus vaccination, suggesting that circulating T cells could not properly focus in the lungs.

Finally, although the efficient formation of the granuloma is not intrinsically protective (26), it is needed to help contain the infection and prevent dissemination and/or subsequent regrowth (27). Models, such as the one presented here, may help to explain how the host response recognizes the presence of a formidable intracellular bacterial infection with the need to induce the formation of a granuloma. This recognition then leads to the formation of a structure consisting of lymphocytes and macrophages rather than a more destructive neutrophil-dominated influx. The evidence to date suggests that the local inflammation and  chemokines stimulate the macrophage influx (23, 28) and that a controlling element is T cells (29), possibly triggered by mycobacterial fragments (30). The current study may have uncovered a previously unrecognized, even earlier-acting mechanism, mediated by the presentation of mycobacterial products by a 2-microglobulin-dependent molecule, that is responsible for the efficient recruitment of lymphocytes directly into the granuloma.