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Infection and Immunity, January 2004, p. 461-467, Vol. 72, No. 1
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.1.461-467.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Urokinase-Deficient Mice Fail To Generate a Type 2 Immune Response following Schistosomal Antigen Challenge

Margaret R. Gyetko,^1* Sudha Sud,¹ and Stephen W. Chensue²

Pulmonary and Critical Care Medicine Division, Department of Internal Medicine,¹ Department of Pathology and Laboratory Medicine, Veterans Affairs Ann Arbor Healthcare System and University of Michigan Medical Center, Ann Arbor, Michigan 48109²

Received 2 June 2003/ Returned for modification 26 July 2003/ Accepted 29 September 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activated lymphocytes express urokinase-type plasminogen activator (uPA). Previous work suggests that uPA modulates T-lymphocyte responses. Mice deficient in uPA (uPA^-/-) fail to generate type 1 (T1) immune responses during infection with Cryptococcus neoformans. Failure to generate either a T1 or a T2 immune response is not predictive of defects in the alternative response. Conversely, down-regulation of one type of immune response may result in inappropriate overactivation of the other. It is not known whether the immune defect in uPA^-/- mice affects only T1 responses or whether T2 responses are also impaired. Impairment of both T1 and T2 responses would suggest a global T-cell defect in the absence of uPA. To determine the role of uPA in T2 immune responses, wild-type (WT) and uPA^-/- mice were primed and challenged with schistosomal egg antigen (SEA). This elicits strong polarization to T2 immune responses in immunocompetent mice. The challenged WT mice developed delayed-type hypersensitivity (DTH) to SEA; high levels of serum immunoglobulin E (IgE); a strong T2 cytokine phenotype with markedly elevated levels of interleukin-4 (IL-4), IL-5, and IL-13; and eosinophil-rich pulmonary granulomas. uPA^-/- mice failed to develop DTH to SEA; did not polarize Ig production to IgE; did not produce high levels of IL-4, IL-5, or IL-13; and had markedly reduced numbers of granuloma-associated eosinophils. uPA^-/- mice fail to generate polarized T2 immune responses to a T2-inducing pathogen. These findings, in conjunction with our previous work, demonstrate that mice deficient in uPA have profoundly impaired immunity involving both T1 and T2 polarization and are largely immunologically unresponsive.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The expression of proteases is thought to mediate the ability of cells to degrade matrix proteins and cross tissue planes during recruitment to inflammatory sites (7, 36, 40). The urokinase-type plasminogen activator (uPA)-urokinase receptor-plasmin system has been implicated as a central mediator in this process (13, 15, 18, 19, 23, 28). Leukocytes express uPA, which converts the inactive proenzyme plasminogen to the broad-spectrum protease plasmin (12). While both mononuclear phagocytes and neutrophils express uPA constitutively, only activated lymphocytes express uPA (4, 14, 29). Thus, in lymphocytes uPA is a strict activation antigen, which suggests that uPA is intrinsically involved in the process(es) by which lymphocytes become activated.

In vivo investigation of the role that uPA plays in pulmonary host defenses has been facilitated by the development of transgenic mice lacking the uPA gene (6). We have demonstrated that uPA is required for protective pulmonary defenses against the fungal pathogen Cryptococcus neoformans (15). A protective host defense against C. neoformans requires the generation of a type 1 (T1) immune response, which is characterized by the cytokines interleukin-2 (IL-2), gamma interferon (IFN-), and IL-12 (21). Wild-type (WT) mice of the background used in this study normally generate protective T1 responses to C. neoformans, clear the infection entirely, and survive. In contrast, uPA^-/- mice have an inability to generate a T1 response in the lung during pulmonary C. neoformans infection. The infection widely disseminates in uPA^-/- mice, and they do not survive. The deficiency of T1 cytokines in the lungs of uPA^-/- mice is due to (i) a decreased number of recruited effector T lymphocytes in the lungs and (ii) a deficiency in T1 cytokine production by uPA^-/- pulmonary mononuclear cells (17). Abundant work investigating the polarization to T1 versus T2 immune responses has clearly demonstrated that defects in one of these types of responses are not predictive of defects in the other but, rather, may suggest overactivation of the unimpaired pathway (8, 10, 39). Therefore, in order to determine how uPA modulates immune responses, it is critical to determine whether only T1 responses are uPA dependent or whether uPA also modulates T2 responses. If T2 responses are also inadequate in the absence of uPA, this would suggest that T lymphocytes require uPA in order to avoid global unresponsiveness, or a state of functional anergy.

To answer this question, uPA^-/- and background-matched WT mice were primed intraperitoneally (i.p.) with Schistosoma eggs and challenged intravenously (i.v.) with schistosomal egg antigen (SEA)-coated beads. This i.v. challenge results in a pulmonary immune response with the formation of discrete intrapulmonary granulomas. When primed WT mice were challenged with SEA, they generated robust delayed-type hypersensitivity (DTH) responses, high levels of immunoglobulin E (IgE). and strong T2 cytokine responses in regional lymph nodes. Additionally, they formed eosinophil-laden granulomas within the pulmonary parenchyma. In contrast, primed uPA^-/- mice failed to develop a DTH, did not polarize immunoglobulin production to IgE, did not produce high levels of critical T2 cytokines, and did not generate granulomas with a large eosinophil component in response to SEA. Thus, uPA^-/- mice have profoundly diminished capacity to generate T2 immune responses compared to WT mice.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals. Mice were housed in specific-pathogen-free isolation rooms in the University of Michigan Department of Laboratory Animal Medicine and the Ann Arbor Veterans Affairs Hospital, both of which are fully accredited by the American Association for Accreditation of Laboratory Animal Care. All procedures were approved by the animal care committees of the Veterans Administration and the University of Michigan Committee on Use and Care of Animals. Mice were periodically checked for murine hepatitis virus and were found to be negative, and they were fed standard animal chow (rodent lab chow no. 5008; Purina, St. Louis, Mo.) and chlorinated tap water ad libitum. Mice were used at 8 to 12 weeks of age.

uPA-deficient mice (uPA^-/-) and background-matched control mice (WT) were generous gifts from Peter Carmeliet. These mice were developed as previously described (6). Genotypes of the uPA and WT mice were confirmed by PCR analysis as described previously (15). Mice of this background (C57B6/129) are immunocompetent and have preservation of complement-dependent acute lung injury (2; E. Angelici, C. Contini, G. Farina, R. Romani, P. Serra, and R. Canipari, Program Abstr. 9th Int. Conf. AIDS, p. 385, abstract no. PO-B10-1502, 1993).

Preparation of SEA-coated beads. Cyanogen bromide-activated Sepharose 4B beads (Sigma, St. Louis, Mo.) were swollen and coupled with SEA (World Health Organization, Geneva, Switzerland) as described previously (8, 9). The total protein coupled was determined to be 4 ng/bead. The beads were stored at 4°C in sterile phosphate-buffered saline (PBS) with 0.05% NaN₃. Beads were washed three times in sterile, preservative-free PBS and counted on a hemocytometer prior to injection.

Induction of immune response to SEA. Mice were sensitized to SEA by i.p. injection of 3,000 Schistosoma mansoni eggs suspended in 0.5 ml of PBS. Fourteen days later, the mice were challenged by tail vein injection of 6,000 SEA-coated Sepharose 4B beads in 0.5 ml of PBS.

Assessment of DTH. Fourteen days following i.p. priming, mice were challenged with injections of 5 µg of SEA in 0.2 ml of PBS in the right footpad and 0.2 ml of PBS in the left footpad. At 48 h after injection, footpad thickness was measured with a micrometer (Lux Scientific Instrument Corp., New York, N.Y.) and expressed in micrometers. The thickness of the right footpad minus the thickness of the left footpad represents the DTH response.

Determination of cytokine and IgE levels by ELISA. At 4 days after bead embolization, mice were killed with an overdose of pentobarbital. Blood was collected, and total serum IgE levels were determined with an enzyme-linked immunosorbent assay (ELISA) kit (PharMingen, San Francisco, Calif.). Regional lymph nodes (hilar and mediastinal) were removed, disaggregated to a single-cell suspension, counted on a hemocytometer, and cultured at 5 x 10⁶ cells/ml in the presence and absence of 5 µg of SEA per ml at 37°C for 36 h. Cytokine concentrations in the conditioned medium were measured (in picograms per milliliter) by ELISA, using specific antibody pairs (PharMingen).

Determination of granuloma volume. Granulomas were measured blindly in hematoxylin- and eosin-stained sections by using a morphometer and a software program (The Morphometer; Woods Hole Educational Associates, Woods Hole, Mass.). A minimum of 20 lesions were measured per lung. The granuloma volume was calculated from the average radii, assuming the lesions to be roughly spherical.

Determination of eosinophil counts within pulmonary granulomas. As a measure of eosinophil mobilization, direct morphometric analysis of eosinophils was performed on five digitized images of granuloma histologic cross sections from the lungs of each mouse as previously described (37). A blinded observer counted eosinophils in six 50-µm² fields from each granuloma.

Statistical analysis. Comparisons between group means were performed by an unpaired Student t test. Where appropriate, data were log transformed to ensure equivalent variances between groups. Statistical calculations were done with StatView 4.5 software (Abacus Concepts, Berkeley, Calif.); n is the number of mice in each experimental group. Data are expressed as means ± standard errors of the means (SEM). Statistical difference was accepted at a P value of 0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Comparison of DTH in uPA^-/- and WT mice. Fourteen days after the mice were primed i.p. with Schistosoma eggs, DTH was determined. As shown in Fig. 1, uPA^-/- mice generated far less DTH than did WT mice (5.50 ± 1.3 versus 21.67 ± 3.97 µm; P 0.003; n = 6). The inability of primed uPA^-/- mice to respond to SEA challenge with appropriate DTH responses suggests that the uPA^-/- mice have impaired T-cell-mediated immune responses to a T2 response-inducing antigen.

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FIG. 1. Comparison of DTH response to SEA in WT (solid bar) and uPA-/- (hatched bar) mice. Primed mice were challenged with footpad injections of SEA. DTH was determined at 48 h. Data are expressed as means ± SEM. *, P < 0.003.

Comparison of IgE production in uPA^-/- and WT mice. A T2 immune response is characterized by an increasing serum IgE level in response to antigen. Therefore, to determine whether mice lacking uPA could generate an appropriate T2 response to SEA challenge, the IgE levels of individual mice were quantified by ELISA (Fig. 2). Unchallenged uPA^-/- and WT mice have comparable low levels of serum IgE (0.099 ± 0.016 and 0.122 ± 0.024 µg/ml, respectively). Following Schistosoma challenge, the level of IgE present in the sera of uPA^-/- mice was significantly less than that seen in WT controls (0.158 ± 0.139 versus 1.983 ± 0.577 µg/ml; P 0.02; n = 5). Thus, the uPA^-/- mice appear to be unable to polarize their immunoglobulin profile to IgE.

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FIG. 2. Comparison of IgE production in WT (solid bar) and uPA-/- (hatched bar) mice. At 4 days following SEA-bead embolization, serum IgE levels were determined by ELISA. Data are expressed as means ± SEM. *, P < 0.02.

Cytokine profiles comparing uPA^-/- and WT mice. Since polarization to a T1 or a T2 immune response is defined by the cytokine profile generated in response to antigen, we compared cytokine production in response to SEA among antigen-primed uPA^-/- and WT mice. Draining lymph nodes were collected from each animal, disaggregated, and cultured at constant cell numbers for 36 h in the presence of SEA. The conditioned media were then collected, and cytokine concentrations were determined by ELISA (Fig. 3). We determined the levels of the T2 cytokines IL-4, IL-5, and IL-13 that were produced. The uPA^-/- mice produced far less IL-4 in response to SEA rechallenge than did the WT mice (uPA^-/-, 164 ± 13 pg/ml; WT, 371 ± 17 pg/ml; P < 0.0001; n = 6) (Fig. 3A). In response to rechallenge with SEA, WT mice produced robust amounts of IL-5 but the uPA^-/- mice produced far less (uPA^-/-, 7,400 ± 834 pg/ml; WT, 19,456 ± 335 pg/ml; P < 0.0001; n = 6) (Fig. 3B). Similar results were found when we quantified IL-13 production. WT mice produced large amounts of IL-13 in response to rechallenge with SEA, whereas uPA^-/- mice produced significantly less (uPA^-/-, 15,460 ± 785 pg/ml; WT, 32,344 ± 1,301 pg/ml; P < 0.0001; n = 6) (Fig. 3C). Thus, the uPA^-/- mice failed to produce appropriate levels of T2 cytokines in response to SEA.

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FIG. 3. Determination of the lymph node cytokine profiles generated by WT (solid bars) and uPA-/- (hatched bars) mice in response to SEA rechallenge in vitro. Regional lymph nodes from mice primed and challenged i.v. with SEA were processed to single-cell suspensions and cultured in the presence of SEA. Cytokine concentrations in the conditioned medium were measured by ELISA at 36 h. Data are expressed as means ± SEM. (A) IL-4. *, P < 0.0001. (B) IL-5. *, P < 0.0001. (C) IL-13. *, P < 0.0001. (D) IFN-. *, P 0.01. (E) IL-2. *, P < 0.0001.

Interestingly, similar results were found when we directed our attention to the classic T1 cytokines IFN- and IL-2. Consistent with Schistosoma being a T2-generating antigen, the overall level of IFN- produced by the draining lymph node cells of all mice was low. Despite this, the level of IFN- produced by uPA^-/- mice was less than that seen in WT mice (uPA^-/-, 124 ± 26 pg/ml; WT, 212 ± 16 pg/ml; P 0.01; n = 5) (Fig. 3D). uPA^-/- mice also produced far lower levels of IL-2 in response to SEA rechallenge than did the WT mice (uPA^-/-, 58 ± 24 pg/ml; WT, 231 ± 16 pg/ml; P < 0.0001; n = 6) (Fig. 3E).

Thus, we see a striking alteration in the elaboration of cytokines in response to schistosomal antigen in uPA^-/- mice. The uPA^-/- mice have a marked inability to elaborate appropriate T2 cytokines in response to antigen. Additionally, uPA^-/- mice produce lower levels of IL-2, a cytokine considered critical for T-lymphocyte competency, and lower levels of IFN-, a T1 cytokine critical to granuloma formation and DTH, than do WT mice. These data demonstrate that the uPA^-/- mice do not aberrantly polarize to a T1 response but rather fail to mount any developed cytokine response.

Comparison of lymph node cell numbers in uPA^-/- and WT mice. Cell-mediated immune defenses are characterized by robust T-lymphocyte proliferation within regional lymph nodes. Grossly, the draining hilar and mediastinal lymph nodes from uPA^-/- mice appeared small compared to the lymph nodes from WT mice. We compared the number of cells collected from the regional nodes of uPA^-/- mice with those from WT mice at 4 days after SEA bead embolization. As shown in Fig. 4, the regional lymph nodes from uPA^-/- mice consistently had fewer cells in response to SEA than did WT mice (P 0.02). Interestingly, despite the reduction in absolute number, the percentages of CD4⁺ cells were comparable in WT and uPA^-/- mice. Thus, in the absence of uPA there is a quantitative deficiency of nodal cells in addition to diminished ex vivo T2 cytokine production when cell numbers are held constant.

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FIG. 4. Comparison of regional lymph node cell numbers in primed and SEA-challenged WT (solid bar) and uPA-/- (hatched bar) mice. Regional lymph nodes (hilar and mediastinal) were processed to single-cell suspensions and counted on a hemocytometer. Data are expressed as means ± SEM. *, P 0.02.

Comparison of eosinophil numbers in pulmonary granulomas in uPA^-/- and WT mice. Eosinophils are generally considered to be terminal effector cells in T2 immune responses, including parasitic infection and allergic responses, and have recently been shown to have an immunomodulatory role in T2 immune responses (24). Pulmonary interstitial granulomas induced by SEA are eosinophil rich (37). In order to assess this component of the T2 immune responses in uPA^-/- mice, the number of eosinophils within pulmonary granulomas of WT and uPA^-/- mice were compared. Six fields of equivalent area within each granuloma were identified (Fig. 5), and the number of eosinophils was counted. uPA^-/- mice formed intrapulmonary SEA-bead granulomas (day 4 postinoculation) that tended to be smaller (by approximately 20%) than the granulomas seen in WT mice, although the difference was not statistically significant (data not shown). Despite this, the pulmonary granulomas of uPA^-/- mice contained approximately half the number of eosinophils seen in the granulomas of WT mice (5.78 ± 0.51 compared to 11.02 ± 1.65; P 0.029; n = 4 or 5 mice) (Fig. 6). Hence, while uPA^-/- mice are able to generate a granulomatous pulmonary response, the granulomas significantly lack the effector cells involved in the modulation of T2 immune responses.

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FIG. 5. Granuloma histology comparing WT (A) and uPA-/- (B) mice (n = 4 or 5). Five random granulomas in hematoxylin- and eosin-stained sections of lungs from WT and uPA-/- mice were chosen. Eosinophils were counted in six 50-µm2 fields from each granuloma. Magnification, x200; inset magnification, x400.

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FIG. 6. Comparison of eosinophil numbers in SEA-induced pulmonary granulomas in WT (solid bar) and uPA-/- (hatched bar) mice. Eosinophil counts determined by morphometric analysis of six 50-µm2 fields of each pulmonary granuloma. Data are expressed as means ± SEM. *, P 0.029.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study demonstrates that uPA^-/- mice have a profound immune defect in response to a T2-eliciting antigen. When uPA^-/- mice are primed and subsequently challenged with SEA they (i) fail to develop a DTH response to SEA; (ii) do not polarize Ig production to IgE; (iii) do not produce high levels of IL-4, IL-5, or IL-13; and (iv) generate pulmonary granulomas that are deficient in eosinophils.

The generation of T2 immune responses during infection with S. mansoni has been well studied experimentally in mice and clinically in humans (11, 31). Like most parasitic helminths, schistosomes elicit strong polarization to the classic T2 cytokines IL-4, IL-5, and IL-13 (9, 31); production of IgE (11, 38); and generation of eosinophil-rich granulomas (37). The Schistosoma egg is the primary antigen that leads to protective T2 immune polarization (35). Interestingly, while a T2 immune response is clearly dominant in response to schistosomal antigens, lower levels of classic T1 cytokines are also produced early in infection and participate in granuloma formation, including the development of DTH reactions (1, 31).

The determination of DTH reactions provides an overall screen for intact cell-mediated immunity, in that intact cognitive (recognition of APC-presented antigens by T cells), activation, and effector phases are required (1). The failure of primed uPA^-/- mice to generate a DTH response to SEA footpad injection demonstrates a defect in specific immunity. The failure of uPA^-/- mice to develop DTH responses is not limited to T2-inducing antigens, as uPA^-/- mice also fail to develop a DTH response to C. neoformans, a T1-inducing antigen (17). The deficit of DTH responses seen in uPA^-/- mice is profound, and the defect in these mice meets at least the clinical definition of an anergic state.

A critical component of host defense against helminths is the development of specific IgE. Schistosomal infection results in large specific and nonspecific IgE responses (30). In vitro, macrophages and other effector cells in conjunction with parasite-specific IgE can become cytotoxic for S. mansoni (22), and human resistance to reinfection has been correlated with specific IgE levels (11). The failure of uPA^-/- mice to produce IgE during schistosomal antigen challenge indicates a failure of specific humoral immunity.

A T2 immune response is defined by the cytokines IL-4, IL-5, and IL-13 (among others). Whereas WT mice produced high levels of these cytokines, the uPA^-/- mice had far lower levels of all of them. The uPA^-/- mice did not polarize to a T2 phenotype. Additionally, the uPA^-/- mice produced less IFN- and IL-2 than did the WT mice, indicating that the uPA^-/- mice did not polarize to the alternative T1 response either. This is in contrast to results of other Schistosoma studies using genetically altered mice. For example, the importance of IL-4 in Schistosoma infection has been shown by using IL-4-deficient mice, which die from severe acute disease. Of interest is that these IL-4-deficient mice did not have less hepatic granulomatous disease (which is partly modulated by T1 cytokines) than the IL-4-replete mice, suggesting that the lethality seen in this model was due to a lack of T2 inhibition of the expression of proinflammatory (T1) cytokines (33). The role of IL-4 in T1-T2 polarization remains controversial, however. In other studies of Schistosoma infection, IL-4-deficient mice not only had reduced production of T2 cytokines (IL-4 and IL-5) but also produced less IFN- (32). IL-4 signals via STAT 6. In a similar Schistosoma model using STAT 6-deficient mice, STAT 6^-/- granuloma cells released IFN- while WT granuloma cells did not. Interestingly, STAT 6^-/- and WT granuloma cells made similar amounts of IL-4 and IL-5. Therefore, STAT 6 limits IFN- production but is not required for production of T2 cytokines (26). By contrast, uPA-deficient mice failed to express substantial levels of either T1 or T2 cytokines in response to SEA.

We have demonstrated that uPA^-/- mice cannot mount protective T1 immune responses against an opportunistic stain of C. neoformans and do not survive infection. The defect in uPA^-/- mice is T-lymphocyte based, because during C. neoformans infection, adoptive transfer of WT immune T cells restores DTH responses in uPA^-/- mice and restores T1 cytokine levels in uPA^-/- mice to the levels produced in WT mice (17). This raises the question as to whether the immune defect in uPA^-/- mice is limited to T1 immune responses or whether the defect is a global T-lymphocyte unresponsiveness, thereby including an inability to generate T2 immune responses. Immune defects in one type of immune response are not predictive of defects in another (38). By contrast, studies have shown that inhibition of appropriate T1 or T2 responses results in excessive or unperturbed expression of the inappropriate, but alternative, immune response, rather than a null response (8, 10, 39). For example, the T1 cytokine IL-12 suppresses the differentiation of T2 lymphocytes, inhibiting the expression of IL-4 and IL-5 and also suppressing IgE production, while up-regulating IFN- expression and stimulating T1 differentiation. In a murine schistosomal model it has been shown that withdrawal of endogenously supplied IL-12 results in rebound increases of IL-4 and IL-13 (41). The striking lack of either a well-polarized T1 or T2 immune response to classic T1- and T2-inducing antigens in the absence of uPA suggests that uPA is required to avoid T-cell unresponsiveness.

Eosinophils are predominant effector cells associated with allergic disease and parasitic infection. Large numbers of eosinophils are recruited to pulmonary granulomas during SEA challenge (37). In addition to their effector function, eosinophils are also important immunomodulatory cells during T2 immune responses, producing IL-4 and stimulating proliferation of antigen-specific T cells (24, 25, 27, 34). Mice deficient in uPA had markedly fewer eosinophils within lung granulomas than did WT mice, indicating a failure of recruitment of a predominant cell in T2 responses, as well as a loss of the eosinophils' immunomodulatory functions. These findings indicate a profoundly blunted T2 immune response in the absence of uPA.

Increasing evidence supports a role for uPA in the modulation of T-cell-mediated host defense. Mice deficient in uPA have impaired pulmonary inflammatory responses and host defense against Pneumocystis carinii in vivo (3). In vitro, T-cell proliferation induced by mitogen or CD3 cross-linking is significantly diminished in the absence of uPA (16). Interestingly, lymphocyte recruitment is unimpaired in uPA^-/- mice, suggesting that the reduced numbers of regional lymphocytes seen during C. neoformans infection or schistosomal challenge are due to diminished T-lymphocyte activation and proliferation (19). Of interest is that two large groups of patients who are particularly vulnerable to opportunistic infections that require T-cell defense (such as C. neoformans and P. carinii infection) are human immunodeficiency virus-infected patients and those who are treated with corticosteroids. PA activity is diminished in alveolar macrophages and in bronchoalveolar lavage specimens from AIDS patients, and free PA inhibitor is increased in AIDS patients with P. carinii infection (2). Similar results were observed in a rat model of corticosteroid-induced immunosuppression and P. carinii infection (Angelici et al., Program Abstr. 9th Int. Conf. AIDS). Corticosteroids are known to inhibit uPA activity (5, 20). Thus, diminished uPA activity appears to be linked to several conditions of T-cell immunodeficiency.

In summary, we show that mice deficient in uPA fail to mount a well-polarized immune response to a T2-inducing antigen. These findings, in conjunction with our previous work (15, 17), demonstrate that mice deficient in uPA are profoundly immunologically hyporesponsive and display characteristics of functional anergy.

 
This work was supported by Merit Research Grants and Research Enhancement Award Program (REAP) funds from the Department of Veterans Affairs (to M.R.G. and S.W.C.) and by National Institutes of Health grants HL60620 (to M.R.G.) and NIAID AI43460 (to S.W.C.). Schistosome materials for this work were supplied through NIH-NIAID contract NO1-AI-55270.

We thank Peter Carmeliet for providing the uPA^-/- and background-matched WT mice and Jeffrey L. Curtis for his thoughtful review of the manuscript.

 
* Corresponding author. Mailing address: 3916 Taubman Center, Medical Center Dr., Ann Arbor, MI 48109-0360. Phone: (734) 761-7980. Fax: (734) 761-7843. E-mail: mgyetko{at}umich.edu.

Editor: W. A. Petri, Jr.

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Infection and Immunity, January 2004, p. 461-467, Vol. 72, No. 1
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.1.461-467.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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