Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Recent studies have emphasized that asthma is an inflammatory airway disease in which T-lymphocytes and eosinophils are critical effector cells (1). In allergic asthma, activated T-lymphocytes within airways express a Th2-like profile of cytokines, including increased secretion of interleukin (IL)-4 and IL-5. These cytokines have been strongly implicated in generating and perpetuating the late-phase asthmatic response, including (1) recruitment of activated eosinophils into airways, (2) airway hyperresponsiveness (AHR), and (3) airflow obstruction (4).

Optimal activation of T-lymphocytes requires two distinct signals. The first signal originates from the ligation of the T-cell receptor (TCR) complex and its co-receptors (CD4 and CD8). The second signal is dependent on "co-stimulatory" signals delivered to T-cells following the interaction of the cell surface molecule CD28 with its counter receptors B7-1 and B7-2 on antigen-presenting cells (7). We, and others, have demonstrated previously that in vivo treatment with soluble CD28 antagonists in animal models can suppress transplant rejection and autoimmunity including diabetes and experimental autoimmune encephalitis (8, 9). Further studies have shown that the inhibitory effects of CD28 antagonists were largely the result of inhibition of IL-2 production and clonal expansion required to generate inflammatory responses. This conclusion was consistent with in vitro studies demonstrating that blockade of CD28 signaling selectively inhibited the activation of Th1 but not Th2 cell clones (10). Thus, conventional wisdom suggested that CD28/B7 interactions are most critical in generating Th1-mediated immune responses.

However, recent studies have shown that differential signaling through the CD28 receptor can have distinct effects on the production of Th2-like cytokines, including IL-4 and IL-5. For example, in vitro differentiation of Th0 cells into Th2, but not Th1, cells was exquisitely dependent upon CD28 stimulation (11, 12). Additionally, we have recently demonstrated that the amount of Th2-like cytokines produced in secondary cultures by antigen-specific TCR transgenic (Tg) T-cells was directly proportional to the intensity of CD28 ligation. Stimulation of TCR Tg+ cells in the presence of the CD28/B7 antagonist, CTLA4Ig, resulted in limited secretion of Th2-like cytokines but normal levels of Th1-type cytokines. This cytokine profile was reproducible when genetically disrupted CD28 knock-out mice were used as the source of T lymphocytes (11). Thus, CD28 signaling can enhance the development of Th2 cells by regulating IL-4 production, but is not essential for the development of Th1 cells. Recent in vivo studies support these in vitro findings. Finck and colleagues showed that in vivo treatment with murine CTLA4Ig inhibited autoantibody production (presumably through inhibition of Th2-like cytokine secretion) and suppressed both the development and ongoing disease in NZB/NZW mice with systemic lupus erythematosus (13). Similarly, Lu and associates found that in vivo CTLA4Ig treatment blocks the Th2-associated mucosal immune response to a nematode parasite (14). Finally, non-obese diabetic (NOD) mice that are CD28-deficient have a higher incidence of disease and increased morbidity compared with CD28-competent animals. This is due to (at least in part) to the inability of CD28-deficient T-cells from NOD mice to produce a protective Th2-like response in the setting of a destructive Th1-mediated autoimmune response (15). Together these data demonstrate that CD28 signaling can promote the differentiation and development of the Th2 lymphocyte subset (7).

In allergic asthma, influencing T-cell differentiation toward the Th1 phenotype might have potential therapeutic application by regulating the effects of eosinophilic inflammation associated with a Th2-like pattern of cytokine secretion in airways. Recent mouse-model studies of asthma have confirmed that blockade of CD28/B7 interactions with CTLA4Ig can inhibit the development of airway inflammation (16). However, these investigations did not elucidate the mechanism(s) by which CTLA4Ig inhibited airway inflammation. In fact, it was suggested that CTLA4Ig did not selectively affect either Th1 or Th2 development in these studies. In the present study, we determined that the ability of the CD28/B7 antagonist, CTLA4Ig, to inhibit the development and progression of airway inflammation and AHR is a direct result of decreased production of IL-4 and IL-5 and increased interferon- (IFN-) production in the respiratory tract of Schistosoma mansoni-sensitized and airway-challenged mice. These data suggest that inhibition of CD28/B7 interactions during antigenic challenge results in concurrent, preferential expansion of Th1-type lymphocytes, and inhibition of the development of Th2-type lymphocytes in this model of allergic airway inflammation.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Animals

Female C57BL/6 mice, 6-10 wk old, were purchased from Harlan Sprague-Dawley (Madison, WI) and housed in a specific pathogen-free facility maintained by the University of Chicago Animal Resources Center (Chicago, IL). The studies reported here conform to the principles outlined by the Animal Welfare Act and the NIH guidelines for the care and use of animals in biomedical research.

Antibodies and Recombinant Proteins

Human CTLA4Ig (a fusion protein comprised of the extracellular domain of CTLA-4 and the Fc portion of the human IgG1 constant region that binds CD28 ligands) was generously provided by Genetics Institute (Cambridge, MA). Anti-CD3 (145-2C11) was produced as previously described (20) and purified by passage over a protein A-coupled sepharose column.

S. mansoni Eggs and Antigen

S. mansoni eggs were isolated, and purified soluble egg antigen (SEA) was produced as previously described (21). Briefly, golden hamsters were infected with about 1,000 cercariae and humanely killed 6 wk later. The livers were removed, placed in phosphate-buffered saline (PBS) containing penicillin (100 U/ml) and streptomycin (100 µg/ ml), and allowed to autodigest for 3 d at room temperature. Next, the livers were homogenized for 3 min and filtered through a stainless-steel mesh to recover the eggs. Eggs were stored at 70°C in 1.7% saline before use.

To prepare SEA, eggs were homogenized on ice in a Tenbroeck tissue homogenizer. Following centrifugation at 10⁶ × g for 2 h, the protein content of the recovered aqueous fraction was determined. SEA was stored at 70°C until used (21).

Antigen Sensitization and Challenge

Our protocol was modified from the procedure of Lukacs and coworkers (22). Mice were immunized intraperitoneally (i.p.) with 5,000 isolated S. mansoni eggs in 0.4 ml saline at Day 0 and challenged with 5,000 additional eggs at Day 7; on Day 14, mice received an intranasal challenge of 10 µg of SEA in 10 µl PBS. Mice were re-challenged intratracheally (i.t.) 7 d later with 10 µg SEA in 25 µl of PBS. A 1-cm midline incision was made in the ventral neck region, and the tissues overlying the trachea were gently separated. Tracheal injection was performed with a 30-g needle. The incision was closed with a single drop of nexabond glue (superglue). Animals were allowed to recover in a warmed humidified incubator. Control animals were sensitized and challenged (SCH) as above with saline instead of eggs and SEA. Anesthesia for this procedure was achieved by i.p. injection of 0.3-0.5 ml of a solution of 9.13 ml PBS, 0.67 ml ketamine-HCl and 0.22 ml xylazine HCl.

Treatment with CTLA4Ig

CTLA4Ig-treated mice were sensitized and challenged in an identical manner as the SCH group. However, beginning on Day 7 the experimental group was injected with CTLA4Ig (50 ug/mouse i.p. in 0.4 cc saline) every other day until death.

Day 0	7	14	21	25
I	----------------I	-----------------I	----------------I	-------------I
eggs (i.p.)	eggs (i.p.)	SEA (nostril)	SEA (i.t.)	death
	CTLA4Ig	-----------------	----------------	--------------

Ventilation and Instrumentation

Four days following tracheal challenge, mice were again anesthetized as described above. The trachea was cannulated with a 20-g, 1-cm metal cannula. The jugular vein was isolated and cannulated with P-10 tubing. The mouse was placed into a Plexiglas volume plethysmograph (see below) and ventilated with 100% oxygen at 140 breaths/ min, tidal volume 0.2 cc (0.2 cc was much greater than the equipment dead space, which was 40 µl).

Measurement of Airway Reactivity

We used a constant-volume, variable-pressure, whole-body plethysmograph (230 ml displacement; Penn-Century, Philadelphia, PA) and Honeywell Microswitch solid-state pressure transducers to measure tidal volume excursions and transpulmonary pressure (Ptp = tracheal cannula pressure minus box pressure, with an open-chest animal). Including the PE-10 tracheal cannula, equipment deadspace is only 40 µl, and equipment resistance is approximately 2.0 cm H₂O/ml/s. Ptp and volume excursions were recorded digitally (500 Hz each), and flow was derived by digital differentiation of the volume signal (Mouse PRC Software; Lakeshore Technologies, Chicago, IL). Lung resistance (R_L) was calculated from these signals breath by breath, by the method of Amdur and Mead (23), after subtracting equipment resistance.

Increasing doses of methacholine (MCh) (44, 133, and 1,200 µg/kg) were infused through the jugular vein catheter at approximately 1-min intervals, at the peak of the response to the preceeding dose. This protocol is a modification of the method of Martin and colleagues (24). In pilot studies, we found that this schedule of MCh administration was sufficient to determine differences in airway reactivity between groups, and minimized the mortality found when higher doses of MCh were used.

Bronchoalveolar Lavage (BAL)

BAL was performed by instilling 0.8 ml of ice-cold PBS through the tracheal cannula, followed by gentle aspiration. This was repeated three additional times. Fluid from all four lavages was pooled. Erythrocytes were lysed with the hypotonic lysing buffer, ACK. Cells were stained with trypan blue to determine viability, and total nucleated-cell counts were established using a Neubauer hemocytometer. Cytocentrifuge preparations were made using a cytocentrifuge (Shandon Southern Instruments, Sewickley, PA) set for 700 × g for 5 min. Cytospin slides were fixed and stained using Diff-Quik (American Scientific Products, McGaw Park, IL). Differential cell counts were determined by counting a minimum of 300 cells/slide, using standard morphologic criteria.

Histology

In some experiments, lungs from mice randomly chosen from all groups were removed from the chest cavity and fixed by injection of 10% buffered formalin (1.0 ml) into the tracheal cannula at a pressure of 20 cm H₂O, and immersed in formalin for 24 h. All lobes were sagittally sectioned, embedded in paraffin, cut in 5-µm sections, and stained with hematoxylin and eosin (H&E) for routine analysis. Five-micron sections were stained by the method of Luna (25) in which Biebrich Scarlet selectively stains eosinophil granules and erythrocytes bright red. Erythrocytes were easily distinguished by size and the absence of granules. Additional sections were stained with Periodic Acid Schiff (PAS) to identify mucin in epithelial goblet cells and submucosal glands. Goblet-cell hyperplasia was graded using a modification of a semi-quantitative scoring scheme (0 = none, 1+ = minimal, 2+ = mild, 3+ = moderate, 4+ = marked) that we have previously reported (26).

Isolation and Stimulation of Lung Lymphocytes

Lungs from individual mice were digested for approximately 1 h in a buffer solution containing 850 U/ml hyaluronidase Type I-S (Sigma, St. Louis, MO), 500 U/ml DNAse-I (Sigma), and 10 mg/ml collagenase (Worthington Biochemical Corp., Freehold, NJ). Undigested tissue was allowed to settle, and the resulting slurry was passed through a 55-µm Nytex filter. Erythrocytes were lysed and the remaining cells were washed 3 times in RPMI-1640 with 10% fetal calf serum (FCS). These washed cells were subsequently overlaid onto a Percoll gradient (50-70%). Cells within the 50-70% interface were aspirated and washed in complete media. An aliquot of these cells was subsequently stained with an anti-CD3 (145-2C11) monoclonal antibody (mAb) and analyzed by fluorescent-activated cell sorter (FACS) to determine relative and absolute numbers of lymphocytes.

From each dissociated lung an equal number of lymphocytes (2 × 10⁵) was suspended in growth medium (RPMI 1640 with 10% FCS, 1% penicillin/streptomycin, 1% glutamine, 0.24% N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), 2 × 10⁵ 2-ME, and non-essential amino acids); placed into 48-well, flat-bottom plates; and challenged with soluble SEA (10 µg/ml) or an irrelevant antigen (ovalbumin 10 µg/ml). Supernatants were removed after 48 h of stimulation and immediately frozen at 70°C until assayed for cytokine content.

ELISA for Determination of Cytokines and IgE in Serum

Cytokines in BAL fluid (IL-4, IL-5, and IFN-), supernatants from lung lymphocytes (IL-5 and IFN-), and IgE antibodies in serum from blood obtained from the jugular vein catheter were detected by commercially available ELISA kits (IL-4 and IFN-, Endogen, Cambridge, MA; IL-5 and IgE, Pharmingen, San Diego, CA).

Statistical Methods

Differences between groups for eosinophils and cytokines in BAL and lung lymphocyte supernatants, and IgE in serum, were determined by Student's t test. Differences between groups for airway reactivity to MCh were determined by ANOVA. Histologic differences between groups for numbers of goblet cells in airway epithelium were evaluated with the Mann-Whitney rank sum test. Statistical analyses of differences for goblet cells were performed using a mean value for each animal. All data are expressed as mean ± SE. Statistical significance was claimed when P  0.05; the power of each test was determined at 0.80 (27).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Airway Reactivity

As seen in Figure 1, S. mansoni SCH mice demonstrated AHR to intravenously administered MCh. These heightened responses were significantly increased at both the 133 and 1,200 µg/kg doses compared with control (3.8-fold increase at the 133 µg/kg dose, P < 0.05; 9-fold increase at the 1,200 µg/kg dose, P < 0.04). To examine the effect of CD28 ligation on the induction of AHR in this model, SCH mice were treated with human CTLA4Ig beginning on Day 7, one week after primary immunization. CTLA4Ig therapy was associated with a profound inhibition of airway responsiveness compared with untreated mice (P < 0.02 versus SCH for 133 and 1,200 µg MCh doses, respectively), and responses of these mice were equivalent to control animals for all three doses of MCh. These results suggest that CD28 engagement was critical for the induction of AHR observed in S. mansoni-sensitized and challenged mice.

View larger version (25K): [in this window] [in a new window]   Figure 1.   CTLA4Ig treatment abolishes the development of airway hyperresponsiveness. Anesthetized and paralyzed mice were intubated, ventilated, and placed in a whole-body volume plethysmograph. Increasing doses of MCh were infused at 1-min intervals through an indwelling jugular catheter. Mice treated with CTLA4Ig (50 µg i.p. every other day beginning at the first antigen challenge; +CTLA4Ig) had airway responses that were equivalent to control animals. These responses were significantly reduced compared with SCH mice (P < 0.02 at 133 µg/MCh and 1,200 µg/MCh). Lung resistance (RL) is expressed as the percentage increase from baseline RL for each group. (In preliminary studies CTLA4Ig had no significant effect on airway responsiveness in unmanipulated mice; data not shown.)

Bronchoalveolar Lavage

Airway inflammation is one of the defining features of asthma, and eosinophils are characteristic inflammatory cells found in asthmatic airways. It has been speculated that eosinophils may release pre-formed cationic proteins and secrete arachadonic acid metabolites that may play an important role in the induction of AHR in asthma (3). In a number of studies of murine models of asthma or humans with naturally occurring asthma, eosinophilic infiltration into airways is dependent on and/or is associated with cytokine production by T-cells with a Th2 phenotype (4, 28- 30). Thus, the presence of eosinophils indirectly reflects both the inflammatory milieu and the T-cell phenotype of the infiltrating cells.

Although eosinophils were not recovered from BAL fluid of any of the control mice (Figure 2), there was a significant number of eosinophils (eos) in BAL fluid from SCH mice (536 ± 85 eos/µl BAL fluid, P < 0.0001 versus control). There was a large degree of variability in the absolute number of eosinophils recovered from BAL of individual mice. However, eosinophils represented, on average, 72% of all BAL cells recovered in the SCH animals. Analysis of the CTLA4Ig-treated group revealed a much different profile. There were significantly fewer eosinophils recovered in BAL from animals treated with CTLA4Ig (209 ± 36 eos/µl, P < 0.001 versus SCH) and these cells represented only 37% of the total cells recovered. These results suggest that CTLA4Ig treatment of S. mansoni-challenged mice dramatically altered the cellular profile of the infiltrating cells.

View larger version (12K): [in this window] [in a new window]   Figure 2.   BAL eosinophil recovery is reduced > 2-fold in mice treated with CTLA4Ig. BAL was performed through a previously placed endotracheal tube. Four 0.8 ml aliquots of normal saline were infused, gently aspirated, and pooled. No eosinophils were recovered in BAL from the control group. Eosinophils recovered from the SCH group represented 72% of all BAL cells recovered. In contrast, eosinophil recovery from mice treated with CTLA4Ig was significantly reduced (P < 0.001 versus SCH) and represented only 37% of total BAL cells recovered in this group. Each circle or square represents a single mouse analyzed. Bars represent the mean and standard error of each group.

Histology

Airways from SCH mice had variable pathologic changes in airway epithelium and submucosa and smooth muscle depending upon the treatment regime. Specifically, eosin-ophilic infiltration and goblet-cell hyperplasia into epithelium and lamina propria was observed in some airways from all SCH mice but no control mice (n = 6). Eosinophilic infiltration and goblet-cell hyperplasia of mucosa were significantly reduced by treatment with CTLA4Ig. Specifically, the mean score for goblet-cell hyperplasia for the SCH group (n = 9) was 2.6 ± 0.2, compared with 1.4 ± 0.3 in the CTLA4Ig-treated mice (n = 9, P < 0.01). There were no significant differences in the size of the airways between groups (mean airway circumference: SCH , 96 ± 2 µm²; CTLA4Ig, 99 ± 2 µm²; P = NS; Figures 3a-3d).

View larger version (137K): [in this window] [in a new window]   Figure 3.   CTLA4Ig treatment inhibits the development of airway tissue eosinophilia and goblet-cell hyperplasia. Lungs from mice from all groups were fixed in situ with 10% formalin for 24 h, processed routinely, and stained with DeLuna to examine eosinophils and with PAS to determine differences in goblet-cell development. B = bronchioles, arrows = vessels. (A) Lung from control mouse. There is no inflammatory infiltrate or edema around bronchioles. Hematoxylin and eosin (H&E) stain, ×200. (B) Lung from SCH mouse. There is a marked, predominantly eosinophilic infiltration and edema around bronchioles and vessels, with extension into the adjacent alveolar parenchyma. H&E stain, ×200. (C) Lung from +CTLA4Ig mouse. There is minimal to mild eosinophilic infiltration and edema within and surrounding bronchioles and vessels. H&E stain, ×200. (D) Higher-power magnification of lung from mouse (B). Profound eosinophilic infiltration of bronchiolar submucosa is evident. DeLuna stain, ×1,000.

Cytokines in BAL

The presence of characteristic Th1- and Th2-type cytokines in BAL fluid was directly evaluated in order to determine the cytokine milieu within airways during SEA challenge. BAL fluid from all three groups of mice was recovered and analyzed for the presence of IL-4, IL-5, and IFN-. The volume of recovered, pooled BAL fluid was equivalent between groups, and ranged from 2.5 to 2.8 ml. As seen in Figures 4 and 5, levels of IL-4 and IL-5 in the BAL fluid of control animals were low, with 10 of 14 samples below the detection limits of our assay (3 pg/ml). In contrast, significant levels of IL-4 (9 ± 2 pg/ml, P < 0.001 versus control) and IL-5 (77 ± 12 pg/ml, P < 0.0001) were observed in BAL fluid from SCH mice. IFN- levels in BAL from SCH mice were equivalent to control animals (0.49 ± 0.05 g/ml control versus 0.66 ± 0.06 ng/ml SCH, P = NS). However, there was a striking increase (approximately 2-fold) in the amount of IFN- (1.19 ± 0.14 ng/ml, P < 0.001 versus SCH) in BAL from CTLA4Ig-treated mice, consistent with an increased expression of Th1-type cytokines in treated animals (Figure 6). Importantly, analysis of BAL fluid from CTLA4Ig-treated animals revealed a significant decrease in the Th2-type cytokines (Figures 4-6). There was an approximately 75% reduction of IL-4 (2.9 ± 1 pg/ml, P < 0.001 versus SCH) and a 50% reduction in the level of IL-5 (46 ± 9 pg/ml, P < 0.04 versus SCH) in BAL fluid from CTLA4Ig-treated mice, consistent with the decrease in BAL eosinophilia observed in these animals. In fact, 77% of the SCH mice had detectable levels of IL-4 in BAL, compared with only 30% of the CTLA4Ig-treated group. This low percentage of IL-4 expression was similar to the percentage of IL-4 expression in BAL of control animals (29%). Similarly, less than 10% of the CTLA4Ig-treated mice had IL-5 levels at or above the mean of the SCH group.

View larger version (12K): [in this window] [in a new window]   Figure 4.   IL-4 content in BAL fluid is significantly reduced in mice treated with CTLA4Ig. Cell-free BAL fluid was analyzed for cytokine content by ELISA. A total 71% of control mice (10/ 14) had IL-4 levels below the limits of detection for our assay (3.125 pg/ml). In contrast, only 36% of the samples from SCH mice (8/22) were below the limits of detection, and IL-4 content of BAL fluid from these mice was significantly increased compared with controls (P < 0.001). Treatment with CTLA4Ig resulted in a significant reduction in IL-4 content compared with the SCH group (P < 0.001); 71% of these samples (15/21) were below the limits of detection for our assay.

View larger version (12K): [in this window] [in a new window]   Figure 5.   IL-5 content in BAL fluid is significantly reduced in mice treated with CTLA4Ig. Forty-seven percent of BAL samples from SCH mice (10/21) had IL-5 levels > 80 pg/ml; the total content of IL-5 in these samples was significantly increased compared with control samples (P < 0.0001). In contrast, only 8% of samples from CTLA4Ig-treated mice (2/24) exceeded 80 pg/ml (P < 0.001 versus SCH). IL-5 content in BAL samples from control mice never exceeded 15 pg/ml.

View larger version (11K): [in this window] [in a new window]   Figure 6.   IFN- content in BAL fluid from CTLA4Ig-treated mice is 2-fold higher than from untreated mice. IFN- levels in BAL fluid from control and SCH mice were equivalent. In contrast, BAL from mice treated with CTLA4Ig had a 2-fold increase in IFN- compared with the SCH group (P < 0.001).

Further analysis confirmed the relative shift in Th1 and Th2 cytokine production. The ratio of IL-4 (pg/ml)/IFN- (ng/ml) in BAL fluid shifted from 12.7:1 in SCH animals to 2.4:1 in the CTLA4Ig-treated group. The ratio of IL-4/ IFN- in BAL fluid from CTLA4Ig-treated mice was almost identical to the control group (2.6:1). Similarly, there was a greater than 3-fold reduction in the ratio of IL-5 (pg/ ml)/IFN- (ng/ml) in BAL fluid of CTLA4Ig-treated animals compared with SCH mice (39:1 versus 116:1, respectively).

Cytokines in Supernatant of Cultured Lung Lymphocytes

Previous studies have shown that multiple cell types, including mast cells, can produce Th2-like cytokines (31- 33). Therefore, we examined directly the cytokine profile of SEA-specific T-cells within lungs. Lung lymphocytes from control, SCH, and CTLA4Ig-treated mice were isolated and stimulated in vitro with SEA (10 µg/ml). Ovalbumin (OVA) (10 µg/ml) was used in additional wells as a control antigen; results of OVA challenge were equivalent to values obtained from media alone and were subtracted from all measurements. Supernatants were harvested at 48 h and analyzed by ELISA for the presence of IL-5 and IFN-. As seen in Figure 7, SEA-stimulated lymphocytes from SCH mice secreted 4-fold elevated levels of IL-5 (1,272 ± 402 pg/ml) compared with T-cells from CTLA4Ig-treated mice (327 ± 125 pg/ml, P < 0.02). Control animals secreted little IL-5 in response to SEA stimulation (< 75 pg/ml). Treatment with CTLA4Ig dramatically and significantly affected IFN- production from in vitro SEA-stimulated lung lymphocytes (Figure 8). Control lung lymphocytes stimulated with SEA produced 0.213 ± 0.07 ng/ml of IFN-. While there was a smallalthough statistically increased amount of IFN- production from SCH mice compared with control mice (0.692 ± 0.205 ng/ml, P = 0.05), there was a dramatic ( 30-fold) increase in IFN- measured from supernatants of CTLA4Ig-treated mice (15.0 ± 7 ng/ml, P < 0.04 versus SCH). These differences were consistent with the BAL cytokine profile of all three groups, and suggested that there was a potent shift from a Th2- to a Th1-type cytokine profile in lung lymphocytes of mice treated with CTLA4Ig.

View larger version (11K): [in this window] [in a new window]   Figure 7.   SEA-stimulated lung lymphocytes from CTLA4Ig-treated mice secrete reduced amounts of IL-5. Lymphocytes recovered in homogenized lungs from randomly chosen mice from all groups were cultured in 48-well plates and stimulated with SEA. Supernatants from control mice secreted little IL-5 in response to SEA (mean value < 100 pg/ml); no IL-5 was recovered in 46% (6/13) of these samples. In contrast, lymphocytes from SCH mice secreted abundant IL-5 (mean value > 1,200 pg/ml); IL-5 was recovered in all wells from SCH mice. Treatment with CTLA4Ig resulted in a 4-fold reduction in IL-5 content compared with SCH (P < 0.02). IL-5 was not recovered in 45% (5/11) of these samples.

View larger version (10K): [in this window] [in a new window]   Figure 8.   CTLA4Ig treatment results in increased secretion of IFN- from SEA-stimulated lung lymphocytes. Supernatants of lymphocytes recovered and treated as above (see Figure 7) from lungs of control and SCH mice secreted relatively little IFN- in response to challenge with soluble SEA. In contrast, SEA-stimulated lymphocytes from lungs of mice treated with CTLA4Ig secreted dramatically increased amounts of IFN-, an approximately 30-fold increase compared with SCH mice (P < 0.03).

Serum IgE

Because IgE levels in serum are dependent upon IL-4 (34) and may be considered an additional index of Th2-cytokine secretion, we measured IgE in serum from animals in all groups. We found that IgE levels in serum from SCH animals (892 ± 186 ng/ml) were significantly increased compared with control mice (308 ± 43 ng/ml, P < 0.02). CTLA4Ig treatment of these mice significantly inhibited the production of IgE (487 ± 45 ng/ml, P < 0.04 versus SCH; Figure 9). These results further support the conclusion that blockade of CD28 ligation suppressed the generation of a Th2-type immune response in this animal model of asthma.

View larger version (11K): [in this window] [in a new window]   Figure 9.   IgE is reduced 2-fold in serum from CTLA4Ig-treated mice. Serum was analyzed for IgE content by ELISA. IgE serum levels in SCH mice were increased almost 3-fold compared with control values (P < 0.02). Treatment with CTLA4Ig resulted in a 2-fold decrease in serum IgE levels compared with SCH mice (P < 0.04).

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The objective of this study was to determine the effect of manipulating CD28/B7 interactions on the development of Th1 and Th2 subsets in a murine model of atopic asthma. We studied S. mansoni-sensitized and airway-challenged C57BL/6 mice because these animals develop Th2-like responses to S. mansoni eggs and soluble antigen as well as airway inflammation that can be inhibited by administration of an anti-IL-4 mAb, suggesting the importance of Th2-type immunity in the development of pathologic changes in airway structure and function observed in this model (22, 35, 36). S. mansoni sensitization and challenge resulted in profound airway eosinophilia that was measurable in airway tissue and in BAL. This was associated with increased airway reactivity to intravenous MCh. Additionally, both IL-4 and IL-5 were increased in BAL fluid and supernatant of lung lymphocytes stimulated in vitro with SEA, and IgE levels in serum were increased almost 3-fold compared with control animals.

Our antigen sensitization and challenge protocol included an important modification to the protocol originally described by Lukacs and associates (22). This original protocol examined airway inflammation and IL-4 within BAL in CBA/J mice from 8-72 h following antigen challenge. In this model, airway eosinophilia and IL-4 content in BAL consistently declined from 48-72 h. Our time-course study in C57BL/6 mice (data not shown) revealed that at 48-72 h after i.t. SEA challenge there was an intense accumulation of eosinophils surrounding vessels. However, at 96 h after antigen challenge eosinophils had migrated from perivascular areas toward airways, so that by 96 h the number of eosinophils within peribronchial spaces was increased compared with any prior time point. Thus, we chose this time to analyze pathologic changes in airway structure and function, and markers for Th1/Th2 cytokine secretion.

One consequence of this change in time points was the decreased levels of IL-4 found in BAL fluid. In this study, the levels of IL-4 in BAL fluid from animals killed at 48 h after antigen challenge were 5-fold higher than the levels of IL-4 in the BAL observed in mice killed 96 h after antigen challenge (data not shown). The relatively low levels of IL-4 in BAL at 96 h may be due to the consumption of IL-4 by the Th2 cells expanding in the ongoing inflammatory process.

We recognize that one potential limitation to interpretation of our data is our use of saline as a negative treatment control (instead of the relevant human Ig) to the huCTLA4Ig-treated group. We and others have previously used huCTLA4Ig to block co-stimulatory pathways in animal models of infectious disease, autoimmunity, transplantation, etc. (37). In previous published studies the more "relevant" control, i.e., Ig, was used and was demonstrated to have absolutely no effect by itself. To our knowledge, in no case was the relevant Ig ever implicated as the possible cause of any important effect. Therefore, we feel confident that our data derived from the huCTLA4Ig-treated group can be compared with our saline-treated negative control group.

We hypothesized that treatment with CTLA4Ig after primary immunization and during the initial antigen challenge period would decrease CD28 signaling at a time when CD28 signaling was critical for enhanced Th2 production (11). Indeed, treatment with CTLA4Ig resulted in greatly diminished airway eosinophilia and normalized reactivity to MCh. Importantly, in these treated animals there was a shift in the Th1/Th2 cytokine profile from a predominant Th2 toward a Th1 pattern in BAL fluid, and supernatant from SEA-stimulated lung lymphocytes. These data demonstrate that CTLA4Ig treatment after sensitization but before antigen challenge leads to increased production of Th1-type, and decreased production of Th2-type, cytokines in airways of S. mansoni antigen-sensitized and airway-challenged mice. These findings support the possibility that Th1/Th2 subset development can be intentionally and concurrently influenced for therapeutic purposes by specific manipulation of CD28 signaling.

The results of our studies on CTLA4Ig-induced Th1/ Th2 deviation differ significantly from studies where CTLA4Ig therapy was initiated at the time of primary immunization. This situation was established using transgenic mice that overexpress CTLA-4 in serum (38). In this study, circulating CTLA4Ig inhibited B7-2-mediated initiation of immune responses by blocking both Th1 and Th2 differentiation. In a follow-up study, this group reported that the number of eosinophils within BAL fluid from Tg mice was greatly reduced. However, the number and phenotype of T-cells from lungs of Tg and normal mice were equivalent, as was the ability of these cells to produce IL-4 and IL-5 upon stimulation in vitro. Specifically, this study concluded that co-stimulation does not induce immune deviation in this model. However, we have previously shown that once established, blockade of CD28 can profoundly alter the Th1/Th2 balance, decreasing the strength of signal and selectively promoting a Th1-like response. Furthermore, asthma is a disease in which the patient has been previously sensitized prior to natural re-exposure to the antigen(s) that causes clinical symptoms. Although our primary interest was in the immunologic consequences of manipulating CD28 signaling after sensitization on the Th1/ Th2 balance, we reasoned that any potential therapeutic interventions of this nature would necessarily be initiated after the patient had experienced a primary antigen exposure. Therefore, we, like others (16), focused our work on the effect(s) of administration of CTLA4Ig after primary immunization with antigen. In fact, under these conditions, we report that CTLA4Ig treatment actually resulted in statistically significant increased IFN- production.

The results of our study imply that CD28 signaling, not necessarily selective ligation of B7-1 versus B7-2, regulates the differentiation and development of the Th1 or Th2 subset of lymphocytes depending upon the strength of signaling and the time of signaling in relation to the kinetics of antigen presentation. This contrasts with other studies that suggest an intrinsic property of the individual B7-1 and B7-2 ligands to preferentially induce the Th1 and Th2 subsets, respectively. In seminal reports, both Freeman and colleagues (39) and Kuchroo and associates (40) suggested that ligation of CD28 to B7-1 versus B7-2 had distinct and separate effects on the promotion of either a Th1 or Th2 phenotype. However, we have performed studies in the mouse model of relapsing experimental autoimmune encephalitis (8) suggesting that the dominant role for B7-1 in this model was a consequence of changes in the expression and function of both B7-1 and B7-2 during the progression of disease. Thus, B7-2 may play a critical role at the time of initial antigen exposure while B7-1 would dominate in the setting of immune responses occurring after antigen exposure (7). This may have particular relevance to asthma, a disease in which random antigen challenge in previously antigen-exposed patients may result in intermittent, up-and-down regulation of both B7 ligands.

At least two fundamental mechanisms have been proposed to explain the effect of inhibiting co-stimulation on T-cell function in vitro and in animal models of human disease. First, blockade of CD28/B7 interactions may inhibit T-lymphocyte activation, effector functions, and the development of both Th1 and Th2 subsets. Alternatively, blockade of co-stimulation may result in immune deviation to skew T-cell effector functions toward either a Th1- or a Th2-like profile. A number of recent studies have attempted to address this issue using murine models of asthma (16- 18). In each of these studies, inhibiting CD28/B7 ligation in ovalbumin-sensitized and challenged mice abolished or inhibited the development of AHR and/or airway eosinophilia. However, the proposed mechanism(s) responsible for these effects were strikingly different in each case. Krinzman and coworkers (17) studied the effect of CTLA4Ig administration prior to sensitization or challenge in BALB/c mice. CTLA4Ig given at either time point resulted in ablation of AHR and inhibition of inflammation. The mechanism(s) underlying these effects was not directly addressed in this study. Keane-Myers and associates (16) also studied the effects of CTLA4Ig administered before either sensitization or antigen challenge. In this study, inhibition of co-stimulation specifically abrogated the production of Th2 cytokines, but had no effect on the development of a population of Th1-type cells. In an additional series of experiments, Harris and colleagues (18) reported the effect of an anti-B7-1 intervention initiated prior to antigen sensitization. The specific effects of this intervention on the development of Th1- versus Th2-like cells within lung were not examined. However, in this experiment B7-1 inhibition had no effect on systemic Th2 responses, and it was concluded that anti B7-1 did not induce immune deviation.

In contrast, our data show that CTLA4Ig administration can result in immune deviation, with a predominately Th2-type pattern manipulated toward a Th1-type pattern. This is consistent with previous work in which we showed exacerbation of autoimmune disease when we bred the NOD mouse to CD28 "knock-outs." This effect was a direct result of a reduced Th2 response and a simultaneous enhanced Th1 response (15). Together, these data suggest that during early stages Th1/Th2 differentiation, increasing the intensity of CD28 ligation promotes a Th2 phenotypic response, whereas blocking these interactions (decreasing "strength of signal") leads to a predominantly Th1 response. Thus, we believe our study demonstrates in an animal model of asthma that CTLA4Ig treatment can promote immune deviation instead of a more global inhibition of T-cell effector function.

In allergic asthma, influencing T-cell differentiation toward the Th1 phenotype might have potential therapeutic application by regulating eosinophilic inflammation associated with a Th2-type pattern of cytokine secretion in airways. In fact, it has recently been speculated that a decrease in childhood exposure to environmental pathogens (including M. tuberculosis) that would normally skew the immunologic response toward a Th1-type pattern has resulted in expansion of Th2-type immunologic responses. This hypothesis has been proposed as an explanation for the striking increase in prevalence of childhood atopy and asthma documented in the United States, Japan, and elsewhere (41).