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Greeshma Somashekar

effect on the immune response to inflammation in the lungs. A model of inflammation in mice was used to determine, spe-cifically, where ITIH4 is expressed in the lung and how it af-fects the response to injury, cytokine counts, and immune cell migration and activation. No other research has been recorded on ITIH4’s role in the lung. Determining the role of ITIH4 allows for further investigation into exactly what is affected. Once we know the aspect of disease that ITIH4 affects and the mechanism behind its actions, we may be able to regulate this process. This would be of significance as it may allow us to, for example, slow the progression of a disease or to inhibit it from attacking the body.

General Background

Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) is a glycoprotein that was discovered in a high-throughput screen-ing assay a little over a decade ago (3).

ITIH4 is one of five heavy chain proteins belonging to the inter-α-trypsin inhibitor (ITI) family. It is the only heavy chain that is not incorporated into an “inter-α-inhibitor com-plex”; rather, it circulates unattached in serum, indicating that it may have a unique function unlike that of other proteins in the family.

Human ITIH4 has been sequenced and a homologous area has been mapped on the mouse genome. In this study, the immune response to inflammatory lung injury was analyzed in ITIH4 knockout (ITIH4-/-) mice and compared to that in wild type (ITIH4+/+) mice of the C57Bl/6 strain. Lipopoly-saccharide, an endotoxin, was used to induce inflammation in mice (8). Inflammation is the initial response to a foreign pathogen by the innate immune system. Cytokines are soluble proteins sent out by damaged tissue that interact with other # cells and induce a state of inflammation in the infected tissue.

Introduction

Rationale and Purpose

In toxicology, a biomarker is a key molecule that links an environmental exposure to a resulting response in the body. These molecules provide clues about the relationship between certain exposures and the development of human disease. Biomarkers are important as they can potentially be used to determine risk, to screen for disease, to do dif-ferential diagnosis, and to monitor disease progression; perhaps their most important function is letting us know that something is out of the ordinary. It is helpful to de-termine the role of a biomarker in order to link it with a particular process or event in disease. A molecule could be a biomarker for anything from cell migration to increased inflammation. If we knew the exact role of a biomarker, its identification would allow us to learn more about the nature of a disease specific to a patient.

Recent studies based on proteomic analysis have identi-fied inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) as a candidate biomarker for several diseases (7). Research-ers arrived at this conclusion after determining that the concentration of ITIH4 is increased relative to baseline levels in patients with certain diseases such as early stage ovarian cancer, breast cancer, and chronic obstructive pul-monary disease (COPD) (1) (6) (9). The protein has never been researched in the lung and its exact function as a bio-marker is unknown. The fact that it persists across several diseases indicates that it may have an important function related to the immune response.

The goal of my project is to better understand the role of ITIH4 as a biomarker of disease. Due to its significance in various diseases, I hypothesized that ITIH4 will have an

Inter-Alpha-Trypsin Inhibitor Heavy Chain 4: Functional Effects on the Inflammatory Response to Lung Injury

Abstract: ITIH4, a serum protein, has been shown to have elevated concentrations in patients with diseases such as early stage ovarian cancer, breast cancer, and chronic obstructive pulmonary disease (COPD). Recent studies have therefore identified ITIH4 as a potential biomarker for these diseases. The goal of this study is to better understand the role of ITIH4 as a bio-marker and specifically, how it affects the immune response to lung injury.Wild type mice (ITIH4+/+) and ITIH4 knockout (ITIH4-/-) mice were exposed to lipopolysaccharide, an endotoxin used to induce inflammation in the lungs. Based on immunohistochemistry, we found that ITIH4 is expressed in the lung in bronchi-al epithelia and in alveolar macrophages and neutrophils. Tissue injury levels, cytokine and cell counts, and cell migration data were analyzed. These indicate that ITIH4 slows the migration of inflammatory cells to the site of an infection. Results suggest that ITIH4 is a biomarker of containment: it serves to localize the immune response to an infection. This information adds to the growing body of knowledge about ITIH4 and may also allow physicians to learn more about the unique immune response in an individual based on the concentration of ITIH4 present.

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tion of physical injury. A Bradford assay done previously in the lab had shown that ITIH4-/- BAL fluid contained less protein than ITIH4+/+ BAL fluid (see Figure 2). To analyze tissue injury qualitatively, sectioned lung tissue from the ITIH4-/- and the ITIH4+/+ mice was stained with H&E and imaged using a Confocal microscope at 10x magnification. Vascular leakage around airways and blood vessels was observed in ITIH4+/+ tissue. This re-sponse was expected in response to moderate endotoxin exposure. In ITIH4-/- tissue, a decreased level of vascular leakage was observed. Since vascular leakage is indicative of protein level, these data support the results obtained previously and suggests that deficient ITIH4-/- mice sus-tain less injury post endotoxin exposure than wild type ITIH+/+ mice, indicating that the presence of ITIH4 increases the immune response surrounding an infection.

KC, MIP-2, IL-6, and TNF-α are inflammatory cyto-kines that we expected to find in our endotoxin-induced model [8. 10]. The infiltration of immune cells such as neu-trophils and macrophages into the infected tissue results in pain and swelling. This research aimed to determine how ITIH4 affects different aspects of the immune response. Results

Our first objective was to establish the presence of our protein of interest, ITIH4, in the mouse lung. Immuno-histochemical characterization was performed on lung tis-sue as well as BAL fluid from ITIH4-/- and ITIH4+/+ mice. Lung tissue slides provided clear images of bronchi-al epithelia, while BAL fluid provided images of inflam-matory cells such as macrophages, neutrophils, and lym-phocytes typically found in alveolar spaces. As expected, there was no staining for ITIH4 in the ITIH4 deficient (ITIH4-/-) mice, and positive staining was observed in ITIH4+/+ mice.

Protein level is often considered a quantitative indica-

Figure 1. Immunohistochemistry for ITIH4: ITIH4-/- mice show no staining for ITIH4 (red) in an airway (A) or on alveolar inflammatory cells (arrowheads; B). Positive stain-ing for ITIH4 was observed in bronchial epithelia (arrows; C) and in macrophages (arrows) and neutrophils (arrows, in-sert) in ITIH4+/+ mice (D). DAPI was used for nonspecific staining of all cells. 40-80x magnification.

Figure 2. Protein Quantification: A. Protein levels are sig-nificantly lower in ITIH4-/- BAL fluid than in ITIH4+/+ BAL fluid.

Figure 4. Cytokine quantification: The concentrations of KC and MIP-2 are significantly higher in ITIH4+/+ BAL fluid (p < 0.0001). Pro-inflammatory cytokines IL-6 and TNF-α, however, had similar concentrations in ITIH4+/+ and ITIH4-/- BAL fluid.

Figure 3. Qualitative Assessment of Injury: B. H&E stain at 10x magnification of lung injury surrounding airways (1) and blood vessels (2) in ITIH4-/- tissue shows mild vascular leakage (arrowheads). C. ITIH4+/+ tissue at 10x magnification shows an increased amount of leakage (ar-rows) suggesting greater injury.

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Having analyzed the effect of ITIH4 on injury, the next step was to determine its effect on the inflammatory re-sponse. BAL fluid from both groups of mice was assayed for four different cytokines (KC, MIP-2, IL-6, TNF-α) using standard ELISA protocol. Pro-inflammatory cyto-kines IL-6 and TNF-α were tested initially due to their proven elevation in response to acute inflammation [1, 7]. However, the presence of ITIH4 made little difference in the concentrations of these two cytokines at the acute phase time point of four hours post exposure. KC and MIP-2 are chemokines that are responsible for the recruitment of inflammatory cells like neutrophils and macrophages to the site of injury. Their concentra-tions are also known to rise post endotoxin exposure [1]. ELISA testing showed that KC and MIP-2 concentra-tions are significantly higher in ITIH4+/+ BAL fluid than in ITIH4-/- fluid (p < 0.0001). The abundance of these chemokines suggests that more inflammatory cells are called to the site of inflammation in ITIH4+/+ mice. This conjecture was tested by counting cells at the same time point.

Cell counts were performed on inflammatory cells from BAL fluid. Results showed that the number of neu-trophils local to the site of injury at four hours post ex-posure was significantly lower (p < 0.0001) in ITIH4+/+ mice. After finding an increased concentration of neu-trophil attractants KC and MIP-2 in ITIH4+/+ mice, this was unexpected. How could there be more signaling molecules calling neutrophils to the site of infection in ITIH4-/- mice, but fewer neutrophils actually present? The cell counts thus were in apparent contradiction with the chemokines quantification results.

Past research indicates that ITIH4 is known to inter-act with actin [5, 6]. Actin plays a role in regulating cell motility. Therefore, it was hypothesized that the pres-ence of ITIH4 may have an effect on cell migration. This would explain the decreased numbers of inflammatory

cells because ITIH4 could be slowing the migration of neutrophils and macrophages to the site of inflammation. This hypothesis was tested by conducting a neutrophil chemotaxis assay. Neutrophil migration assay results showed that ITIH4 does, in fact, inhibit cell motility. The greatest level of migration was observed in ITIH4-/- cells through ITIH4-/- serum while the least migration was observed in ITIH4+/+ cells through ITIH4+/+ serum (p < 0.001). When either the serum or the cells were ITIH4 defi-cient, intermediate results were observed. The difference was statistically significant even after correction for multiple comparison testing.

To test whether our observations were limited to a spe-cific lung injury model or cell type, differential cell counts were also done on ITIH4+/+ and ITIH4-/- BAL fluid three days after bleomycin exposure and three days af-ter naphthalene exposure. Bleomycin is the product of a fungus and exposure is known to induce intratracheal inflammation and fibrosis (scarring) on a time course of 3 – 21 days post exposure. Naphthalene is known to in-duce temporary airway injury in epithelial cells [7]. Neu-trophil levels were significantly higher in ITIH4-/- mice (p < 0.05) post bleomycin exposure while macrophage lev-els were significantly higher in ITIH4-/- mice (p < 0.01) post naphthalene exposure. These results indicate that the effect of ITIH4 is nonspecific to a particular type of cell or injury.

ConclusionBased on immunohistochemistry, we determined that

ITIH4 is expressed in bronchial epithelia and alveo-lar macrophages and neutrophils. Lung injury, protein levels, and chemokines levels were lower in ITIH4-/- mice

Figure 5 (above). Cell Differentials: Neutrophil counts are significantly lower in ITIH4+/+ BAL fluid (p<0.0001). However, macrophage and lymphocyte counts are only slightly lower in ITIH4+/+ BAL fluid and were not statistically significant.

Figure 6. (below). Neutrophil in vitro migration assay: Migration is higher in ITIH4-/- serum than in ITIH4+/+ serum. Also, cells deficient of ITIH4 migrate faster than cells with ITIH4. Results were statistically significant (p < 0.05) in both cases.

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than in ITIH4+/+ indicating that the presence of ITIH4 promotes the inflammatory response at the site of infection. Additionally, total cell and neutrophil counts are elevated in ITIH4-/- mice relative to the ITIH4+/+ mice. This is ex-plained by migration assay data, which indicates that ITIH4 presence inhibits migration. To summarize: ITIH4 promotes the immune response but slows immune cell migration post endotoxin exposure in the lungs.

We found that ITIH4 is expressed in bronchial epithelia and in alveolar macrophages and neutrophils, which led to questions about its function. Additional results indicate that ITIH4 plays a role in increasing the inflammatory response at the site of infection. It simultaneously slows the migration of inflammatory cells to the affected area. The acute phase immune response is a crucial bodily defense against infection. In many diseases such as COPD and various cancers, the initial immune response could cause greater damage than the disease itself. Since the concentration of ITIH4 is elevated in these diseases, ITIH4 may serve to prevent the body from overreacting to infections. This information is a significant addition to the current body of knowledge about ITIH4, be-cause we are able to better understand its role as a biomarker.

Based on collected data, we believe that ITIH4 may be a containment protein. The fact that ITIH4 promotes the inflammatory response at the site of infection while slow-ing the migration of immune cells suggests that its primary purpose is to localize the immune response to a specific re-gion. Often, a large scale immune response is more danger-ous than the infection itself. Therefore, ITIH4 may prevent the immune response from proliferating at a rate that would cause more harm than good. At the same time, it contains the response within a reasonable area, preventing the infection from spreading too quickly.

The nature of the interaction between ITIH4 and actin is unknown. Do the two bind directly or are other molecules and proteins involved? Immunoprecipitation can be used to find the binding partners of isolated ITIH4 to determine the physical nature of the bond. All cell migration

assays can be repeated with human neutrophils and macrophages in the future. This is a pending transla-tional study. Human ITIH4 can be purified, ensuring that ITIH4 is the only molecule causing the differences in migration, and can be tested at varying concentra-tions. The concentration gradient has the potential to suggest a biological range for humans in which the pres-ence of ITIH4 affects the immune response to infec-tion and cell migration. ITIH4 is known to be elevated in sera of patients with many cancers [4] and therefore its effect on cell migration may extend to cancer me-tastasis. This would be an interesting area to look into. ITIH4 is known to have three protein domains: the vault domain (VLT), the von Willebrand domain (VWA), and the multi-copper oxidase domain (MCOD). VLT and VWA are homologous to all members of the ITI family, while MCOD is unique to ITIH4. VWA is known to bind to integrins and to promote adhesion, while MCOD plays a role in iron metabolism [3, 4, 5]. These known functions allowed us to make predictions about the roles of these three domains in ITIH4: VWA may affect im-mune cell migration and MCOD may affect immune cell activation.

Future work will focus on finding a correlation between certain domains and effects on immune cell activation and migration. Focusing our research on specific domains will allow us to determine the mechanisms behind ITIH4’s role as a biomarker.

To test whether our observations were limited to a spe-cific lung injury model or cell type, differential cell counts were also done on ITIH4+/+ and ITIH4-/- BAL fluid three days after bleomycin exposure and three days after naphthalene exposure. Bleomycin is the product of a fun-gus and exposure is known to induce intratracheal inflam-mation and fibrosis (scarring) on a time course of 3 – 21 days post exposure. Naphthalene is known to induce tem-porary airway injury in epithelial cells [7]. Neutrophil lev-els were significantly higher in ITIH4-/- mice (p < 0.05) post bleomycin exposure while macrophage levels

Figure 7. A. Cell differential count three days post bleomycin exposure indicates an increase in neutrophils in ITIH4-/- mice. B. Three days post naphthalene ex-posure, there is also an increase in macrophages in ITIH4-/- mice.

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Acknowledgements

Stavros Garantziotis, Principal Investigator - Matrix Biology Group, NIEHS Vandy Stober, Lab Manager – Matrix Biology Group, NIEHSMyra Halpin, North Carolina School of Science and Math-ematics

References

[1] Bandow et al, 2008. Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies – COPD biomarker discovery study. Proteomics 8:1-11.[2] Bhanumathy et al, 2002. Itih-4, a serine protease inhibitor regulated in interleukin-6-dependent liver formation: role in liver development and regeneration. Developmental Dynamics 223:59-68. [3] Cai et al, 1998. Identification of mouse itih-4 encoding a glycopro-tein with two EF-hand motifs from early embryonic liver. Biochimica et Biophysica Acta 1398:32-37.[4] Choi-Miura et al, 2000. The novel acute phase protein, IHRP, in-hibits actin polymerization and phagocytosis of polymorphonuclear cells. Inflammation Research 49:305-310.[5] Kroczynska et al, 2005. BIP co-chaperone MTJI/ERDJI interacts with inter-α-trypsin inhibitor heavy chain 4. Biochemical and Biophysi-cal Research Communications 338:1467-1477.[6] Mohamed et al, 2008. Lectin-based electrophoretic analysis of the expression of the 35 kDa inter-α-trypsin inhibitor heavy chain H4 frag-ment in sera of patients with five different malignancies. Electrophoresis 29:2645-2650.[7] Pineiro et al, 1999. ITIH4 serum concentration increases during acute-phase processes in human patients and is up-regulated by inter-leukin-6 in hepatocarcinoma hepG2 cells. Biochemical and Biophysical Research Communications 263:224-229. [8] Rojas et al, 2004. Endotoxin-induced lung injury in mice: structural, functional, and biochemical responses. Am J Physiol Lung Cell Mol Physiol 228:L333-L341.[9] Tang Y et al, 2008. Progenitor/stem cells give rise to liver cancer due to aberrant TGF-β and IL-6 signaling. PNAS 105: 2445-2450. [10] Wohlford-lenane et al, 1999. Cytokine gene expression after inhala-tion of corn dust. The American Physiological Society 99:L736-L743.

were significantly higher in ITIH4-/- mice (p < 0.01) post naphthalene exposure. These results indicate that the effect of ITIH4 is nonspecific to a particular type of cell or injury.

Materials and Methods

AnimalsRight lung tissue and bronchoalveolar lavage (BAL) flu-

id samples were provided by a contract laboratory. Samples were extracted from ITIH4-/- and ITIH4+/+ mice of the C57Bl/6 strain. All mice had been exposed to endotoxin (5 ml of 1 mg/ml endotoxin solution was added to 200 ml phosphate buffered saline) via a Collision nebulizer for 2.5 hours.

Cytokine Quantification in Lavage FluidCommercially prepared ELISA kits (R&D Systems

Duoset, Minneapolis, MN) were used to measure the con-centrations of murine cytokines (KC, MIP-2, IL-6, and TNF-α) in BAL fluid as per the manufacturer’s provided instructions.

Differential Cell CountsBAL fluid aliquots containing alveolar cells were spun

onto slides using Cytospin 3 (Shandon, Pittsburgh, PA). Neutrophil, macrophage, and lymphocyte counts were de-termined after Wright-Giemsa staining.

Histology and Immunohistochemical Characteriza-tion

Lungs were inflated with 10% formalin and embedded in paraffin. Five micrometer sections were obtained from the central portion of the lungs. Slides were stained either with hematoxylin and eosin (H&E) or with rabbit poly-clonal anti-mouse ITIH4 antibody. Images were produced using a Zeiss 510 NLO Confocal microscope (Zeiss, Pe-tersburg, VA).

Neutrophil IsolationBone marrow isolated from ITIH4-/- and ITIH4+/+

mice was stored in Hank’s balanced salt solution (HBSS). Cells were centrifuged over a 52/64/72% Percoll gradient. Purified neutrophils were removed from the layer between the 52% and 72% bands.

Neutrophil Chemotaxis AssayThe chemoattractant MIP-2 was added to the lower

chambers on a 24 transwell plate. Isolated neutrophils were added to the upper chambers. The plate was incubated for 1 hour at 37 °C. After incubation, cells in the lower chamber were counted to quantify neutrophil migration. ITIH4-/- or ITIH4+/+ cells were incubated in ITIH4-/- or ITIH4+/+ serum to create a 4x4 template.

Macrophage Migration AssayCells harvested from extracted bone marrow were placed

in media. Media was then removed and adherent cells were washed with prewarmed PBS. Macrophages were trypsin-ized using 0.25% trypsin-EDTA as per

standard tissue culture protocol. The solution was centri-fuged to pellet cells and resuspended in 1 ml of media for cell counts. The cell concentration was then adjusted to 1 x 106 cells/ml and incubated at 37 °C for 7 days. The chemoattrac-tant fLMP was added to the lower chambers on a 24 tran-swell plate. Cultured macrophages were added to the upper chambers. A similar template was used as for the neutrophil assay shown above, with fLMP replacing MIP-2 as the at-tractant. The plate was incubated for 2 hours at 37 °C. Cells were washed, fixed in 2% paraformaldehyde, and stained with DAPI. Transwell filters were removed and placed on slides, which were imaged. Cell counts were performed to quantify macrophage migration.