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The Journal of Immunology, 2002, 169: 1801-1809.
Copyright © 2002 by The American Association of Immunologists

The Th1-Specific Costimulatory Molecule, M150, Is a Posttranslational Isoform of Lysosome-Associated Membrane Protein-11

Durbaka V. R. Prasad*, Vrajesh V. Parekh*, Bimba N. Joshi*, Pinaki P. Banerjee*, Pradeep B. Parab*, Samit Chattopadhyay*, Anil Kumar{dagger} and Gyan C. Mishra2,*

* National Centre For Cell Science, Pune, India; and {dagger} School of Biotechnology, Devi Ahilya Vishwavidyalaya, Indore, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4+ T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4+ T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hallmark of the adaptive immune system is its ability to induce as well as regulate its responses to a given infectious agent. Activation of CD4+ T cells minimally requires two distinct signals to be provided by an APC. Although the first signal is provided by the MHC class II molecule presenting the appropriate Ag-derived peptide, the second signal is delivered by a class of accessory molecules that are also known as costimulatory molecules (1). A number of such costimulatory molecules have now been discovered, along with their counterreceptors on T cells. The most prominent ones include B7.1 and B7.2 that interact with CD28 on T cells (2, 3, 4), OX40 ligand-OX40 (5, 6), 4-1BBL-4-1BB (7, 8), and B7RP-1 that bind to inducible costimulatory (9, 10). On the basis of their ability to secrete specific lymphokines, CD4+ T cells have been divided into Th1 and Th2 subsets. Th1 lymphocytes contributing to cellular immunity are characterized by the production of IL-2 and IFN-{gamma}, whereas Th2 lymphocytes produce IL-4, IL-5, IL-10, and IL-13, which are thought to be mainly involved in humoral immunity (11, 12). Th1- and Th2-associated cytokines tend to be reciprocally regulatory. IFN-{gamma} inhibits Th2-associated functions (13), while IL-4 and IL-10 have negative effect on Th1-associated functions (14). It has been postulated that these two helper cell subsets are not only functionally different, but also show qualitative and quantitative distinctions in their requirements for costimulation (15).

In addition to ensuring the activation of T cells, accumulating evidence suggests that costimulatory molecules may also play a role in regulating the qualitative aspects of T cell responses. For example, at least when expressed on activated B lymphocytes, B7.1/CD28 has been shown to predominantly activate the Th1 subset of CD4+ T cells (16, 17, 18), whereas B7.2/CD28 appears to bias toward Th2 responses (19, 20, 21). Similarly, 4-1BB-4-BBL interactions preferentially contribute toward the development of Th2 responses. The recently identified costimulatory molecule B7RP-1, which is known to induce IFN-{gamma} production, is widely expressed on B cells and macrophages (10). In this context, a new costimulatory molecule, B7-DC, which is specifically present on dendritic cells, has also been described. This molecule induces Th1-specific polarization (22).

In an earlier study, we have reported the isolation of a 150-kDa protein (M150) from the surface of activated macrophages. This protein was shown to possess costimulatory activity, and was capable of stimulating T cell proliferation. In addition, it was also able to induce secretion of lymphokines that are typical of Th1 responses (23). Furthermore, we showed that macrophages predominantly use M150, relative to B7.1 for costimulating proliferation and IFN-{gamma} production from Th cells (24). M150 was also shown to restore normal Th1 function upon bystander costimulation in diseases like leishmaniasis and tuberculosis, where Th1-like responses are supressed (25, 26). However, the biochemical identity of M150 has not been elucidated so far.

In the present study, we identify M150 as a specific posttranslational isoform of constitutively produced lysosome-associated membrane protein-1 (LAMP-1).3 Interestingly, the costimulatory activity of this molecule depends upon its unique pattern of glycosylation that is generated only in activated macrophages. Thus, M150 represents a novel example of a housekeeping protein that can also function as a costimulatory molecule, and adds to the expanding list of APC-restricted costimulatory molecules with a biased influence on T cell function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Macrophage membrane preparation and purification of M150

The peritoneal exudate cells were harvested from BALB/c mice (6–8 wk old) injected 4 days previously with 2 ml of 3% thioglycolate (Difco, Detroit, MI). The peritoneal exudate cells were washed with cold HBSS and macrophages were obtained by adhering for 1 h at 37°C on plastic petri dishes and the purity of macrophage was >98%, as analyzed by their reactivity with anti-mac-1 Ab. The macrophages were washed thrice and pellet was frozen overnight at -70°C. The cells were thawed and homogenized in the presence of 20 mM Tris-Cl (pH 7.4) and 1 mM EDTA along with protease inhibitor mixture (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml antipain, 10 mM iodoacetamide, 10 µg/ml chymostatin, and 1 mM PMSF). The nuclear fraction was removed by centrifuging at 700 x g for 10 min at 4°C. The supernatant was subjected to centrifugation at 110,000 x g for 2 h at 4°C. The pellet was solubilized overnight in 20 mM Tris-Cl (pH 7.5) containing 1% Triton X-100, 20% glycerol and protease inhibitor mixture, and recentrifuged at 100,000 x g for 1 h at 4°C to remove the insoluble debris. The membrane proteins (27, 28) in the supernatant were separated on 10% SDS-PAGE (29). M150 was eluted from the gel by crushing the gel pieces containing the protein band followed by overnight incubation with 100 mM NH4 HCO3, 50 mM Tris-Cl, 0.1 mM EDTA, and 150 mM NaCl (pH 8.0). SDS from the protein solution was removed by passing through Extracti-D gel column (Pierce, Rockford, IL) and the protein content was estimated using the bicinchoninic acid kit (Pierce). The protein was further purified by fast performance liquid chromatography on Mono-Q column and the purity was ascertained by two-dimensional electrophoresis before subjecting it to protein sequencing.

Sequencing of M150

The internal and amino-terminal sequencing were conducted independently at two different facilities. Typically, the purified M150 was electrophoresed on 10% SDS-PAGE and blotted on to polyvinylidene difluoride membrane using 10 mM CAPS buffer (US Biochemicals, Cleveland, OH) containing 10% methanol at 200 mA for 2 h at 4°C. The blot then was subjected to tryptic digestion and the peptides thus obtained were sequenced for internal sequencing at the protein sequencing facility of Worcester Foundation for Experimental Biology. The protein obtained by FPLC was subjected to N-terminal sequencing at the sequencing facility of Purdue University (West Lafayette, IN).

Heteroduplex analysis

Primers were designed from the internal stretches of the M150 protein that was sequenced earlier. Sense primer 5'-GAGATCTACACAATGGACTC-3' and antisense primer 5'-GAGTCCATXGTGTAGATCTC-3' were custom made (Life Technologies, Grand Island, NY). The cDNA of LAMP-1 cloned from mouse embryo 3T3 cDNA library was a gift from Dr. J. T. August (John Hopkins University, Baltimore, MD). PCR was performed using these primers at an annealing temperature of 55°C. RT-PCR was done using the same primers from RNA isolated from thioglycolate-elicited macrophages using S.N.A.P. total RNA isolation kit (Invitrogen, San Diego, CA). A total of 500 ng of total RNA was used to perform RT-PCR using Titan one tube RT-PCR system (Roche, Indianapolis, IN). The product was subcloned in PCR cloning vector. After transformation and plating from several different colonies, PCR was performed from plasmid preparations using the same primers. A total of 0.5 µg of each PCR product was mixed with 0.5 µg of PCR product obtained from the cDNA construct of LAMP-1. Heteroduplex analysis was conducted in TNE buffer, final concentration Tris:NaCl:EDTA equals 1/10/0.1 mM (30). The PCR product mixtures were made to a final volume of 10 µl and heated at 95°C for 5 min followed by rapid cooling in an ice bath for 60 min. The sample was mixed with loading dye and electrophoresed on an 8% native PAGE using TBE buffer. DNA pattern was visualized by ethidium bromide staining.

mAb preparation

Lewis rats 8–10 wk old, obtained from National Institute of Immunology (New Delhi, India) were immunized by i.p. injections of thioglycolate-elicited macrophages (107 cells/mice) from BALB/c mice. Three booster doses were given after 21 days of primary immunization at an interval of 2 wk each. The spleen was removed on the third day after the final booster dose and the cells were fused to SP2/01-AG14 (American Type Culture Collection, Manassas, VA) using PEG 1500 (Roche). Hybrids were selected in hypoxanthine/aminopterin/thymidine medium (Life Technologies). The supernatants were screened for M150 reactivity both by ELISA and Western blot analysis. Positive clones were subcloned by limiting dilution and the isotype of Abs secreted by individual clones was determined using mAb-based Rat Ig isotyping kit (BD PharMingen, San Diego, CA). For the present study, anti-M150 Abs were produced as ascites, and purified by sequential chromatography over Sepahcryl S 300, followed by goat anti-rat IgM affinity chromatography.

FACS analysis

The affinity-purified mAb (G1) against M150 and control IgM (normal rat IgM, affinity purified by sheep anti-rat IgM; Pierce) were FITC conjugated. PE-conjugated anti-Mac-1, anti-B220, anti-CD4, anti-CD8, normal IgG2a, normal IgG 2b, and FITC-labeled anti-LAMP-1 Ab, FITC-labeled normal rat IgG2a (isotype control for anti-LAMP-1), and Fc block were purchased from BD PharMingen. Splenocytes and macrophages (106 cells), either resting or activated with IFN-{gamma} (5 ng/ml for 12 h) from C57 BL/6 mice, were first incubated with Fc block for 20 min at 4°C and the splenocytes were then dually stained either with FITC-labeled anti-M150 or anti-LAMP-1 along with various PE-conjugated CD markers. Incubations were done at 4°C in FACS buffer (2% FCS, 0.5% BSA in PBS, pH 7.2) and washed using the same buffer three times before incubation with the secondary Ab. Finally, cells were washed and fixed in 1% paraformaldehyde in PBS and staining was analyzed by flow cytometry (FACSVantage; BD Biosciences, Mountain View, CA). Macrophages were used for the competitive binding experiment between the anti-LAMP-1 and anti-M150 Abs.

Western blotting

The samples were electrophoresed using 10% SDS-PAGE (SE 260; Amersham Pharmacia Biotech, Uppsala, Sweden) and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using 20 mM Tris containing 125 mM glycine (pH 7.5) and 20% methanol at 200 mA for 2 h at 4°C. The membrane was blocked with 2% BSA in TBST (50 mM Tris-Cl, 150 mM NaCl, 0.05% Tween 20, pH 8). Incubations and washings of primary and secondary Abs were done with TBST at room temperature. Blots were developed using diaminobenzidine (Sigma-Aldrich, St. Louis, MO).

Generation of LAMP-1-Fc fusion protein

The pCEP4-hFc vector that encodes the Fc portion of human IgG1 was a gift from Drs. T. W. Mak and Dr. S. K. Yoshinaga (Amgen, Thousand Oaks, CA). For cloning LAMP-1 in pCEP4-hFc vector, 370 aa from the amino-terminal of LAMP-1 were fused in frame to the sequence encoding the Fc portion in the amino terminus region. The forward and reverse primers, 5'-CGCAAGCTTATGCGGCCCCCGCGCGCG-3' and 5'-CGCGCGGCCGCGTTGTTACCATCCTGAACACACTC-3' were used for cloning the LAMP-1 truncated gene into the HindIII and NotI sites within the multiple cloning sites of pCEP4. The coding sequence of LAMP-1 from N terminus up to the transmembrane domain, devoid of the region spanning the membrane, was incorporated into pCEP4-Fc. The highly purified pCEP4-LAMP-1-Fc plasmid was then transfected into Chinese hamster ovary (CHO) cells and the mouse macrophage cell line P388D1 using FuGene 6 transfection reagent (Roche). The transfected cells were grown in serum-free media containing insulin-transferrin-selenium-A (Life Technologies). Soluble secreted fusion proteins were purified from culture supernatants using protein A agarose affinity column chromatography (Roche).

Deglycosylation of transfected fusion proteins

A total of 5 µg of fusion proteins, i.e., CHO-LAMP-1-Fc or macrophage-LAMP-1-Fc, were treated with 20 mU endoglycosidase H (Roche) for 16 h at 37°C using 0.5 M sodium citrate buffer (pH 5.5) containing 0.1 M 2-ME and 0.1% SDS.

T cell proliferation, cytokine, and blocking assays

Spleens from 6- to 8-wk-old female BALB/c obtained from experimental animal facility of our institute were used to make single-cell suspensions of splenocytes. RBCs were lysed using hemolytic Gey’s solution. Nonadherent cells were collected from supernatants after allowing cells to adhere to plastic petri dishes (Corning Glass, Corning, NY) at 37°C in the presence of 5% CO2 for 2 h. The CD4+ T cells were enriched by passing through a nylon wool column (Robbins Scientific, Sunnyvale, CA). Finally, CD4+ T cells were purified to at least 98% purity by negative selection using the CD4+ T cell enrichment mixture (StemCell Technologies, Vancouver, British Columbia, Canada). The T cells were cultured in RPMI 1640 (Life Technologies) supplemented with penicillin (70 µg/ml), streptomycin (100 µg/ml), glutamine (4 mM), 2-ME (50 mM), sodium pyruvate (1 mM), HEPES (20 µM), and heat-inactivated 10% FCS (Life Technologies). The purified T cells (105) were cultured along with anti-CD3, either in presence or absence of varying concentrations of Fc fusion proteins for 72 h in 96-well, flat-bottom plate (Costar, Cambridge, MA). These cells were then pulsed with [3H]thymidine (1 µCi/well) during the final 12 h of the culture period, following which the incorporated radioactivity was determined by liquid scintillation counting. For blocking experiments, anti-LAMP-1 (ID4B) and IgG2a isotype (BD PharMingen), along with affinity-purified anti-M150 and normal rat IgM were used. In parallel experiments, supernatants from such cultures were also collected for determination of IL-2, IFN-{gamma}, IL-4, and/or IL-13 by ELISA (R&D Systems, Minneapolis, MN).

RT-PCR for c-Maf and T-bet

Total RNA was prepared from 106 CD4+ T cells per well cultured in 24-well plate for 24 h either in the presence of CHO-LAMP-1-Fc or macrophage-LAMP-1-Fc and 1 µg/ml of soluble anti-CD3 (BD PharMingen). The total RNA was isolated using S.N.A.P. total RNA isolation kit (Invitrogen). A total of 500 ng of total RNA was used to perform RT-PCR using Titan one tube RT-PCR System (Roche). The T-bet primers used were 5'-ATGGGCATCGTGGAGCCGGGCT-3' and 5'-ACTTGGACCACACAGGTGGTTG-3', cMaf primers were 5'-ACTGAACCGCAGCTGCGCGGGGTCAG-3' and 5'-CTTCTCGTATTTCTCCTTGTAGGCGTCC-3' (31), which were custom made (Gemini Biotech, Alachua, FL). The control primers used were dihydrofolatereductase set for RT-PCR (Stratagene, La Jolla, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The protein M150 displays amino acid sequence homology with LAMP-1

In our earlier studies, we have isolated a 150-kDa protein from the membrane fraction of activated murine macrophages. This protein, termed as M150, was demonstrated to provide costimulatory activity to CD4+ T cells. Interestingly, expression of M150 on the surface of the activated macrophages was found to specifically elicit Th1 responses from CD4+ T cells, at least when assessed at the level of cytokine production in culture supernatants. Furthermore, surface expression of M150 could be detected on activated macrophages but not on activated B cells. Thus, M150 constitutes a macrophage restricted costimulatory molecule that specifically drives Th1 responses. Therefore, it was of interest to establish the biochemical identity of this cell surface protein.

Using a previously established procedure, a homogenous preparation of M150 was obtained from the plasma membrane fraction of activated macrophages (Fig. 1Go, A and B) and the purified M150 was subjected to internal and amino-terminal sequencing by two independent preparations at two different sequencing facilities. The three partial sequences thus obtained were subsequently used in a search for homologous sequences within the protein databases. Surprisingly, all of the three partial sequences yielded 100% identity with segments of the murine LAMP-1 protein. As seen in Fig. 1GoC, the amino terminus of M150 was identical with that of LAMP-1, and the two internal sequences obtained were found to correspond to segments between aa 128–139 and 288–293 of the LAMP-1 protein. These results strongly suggested that M150 is either identical with, or highly homologous to LAMP-1 (32, 33).



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  		FIGURE 1. Electrophoretic analysis of M150 protein and its characterization. A, SDS-PAGE of plasma membrane proteins isolated from thioglycolate exudate macrophages. Macrophage membrane proteins were prepared as described in Materials and Methods. In brief, macrophages (98% mac-1 positive) were homogenized and after removal of the debris by low-speed centrifugation, the supernatant was subjected to ultra centrifugation at 110,000 x g. The pellet was solubilized and it was recentrifuged at 100,000 x g. The supernatant containing the membrane fraction was separated on a 10% SDS-PAGE and was stained with Coomassie blue. Lane 1, Molecular mass markers; lane 2, Surface membrane proteins of macrophage; lane 3, Gel-purified M150 under reducing condition. B, Two-dimensional electrophoresis of M150. The purified M150 (as shown in A, lane 3) was further purified by fast performance liquid chromatography on a Mono-Q column and subjected to two-dimensional electrophoresis. The M150 was resolved by isoelectro focusing across a pH gradient of 3.5–9.5 (first dimension) and then electrophoresed on a 10% SDS-PAGE (second dimension). C, Sequence identity of partial amino acid sequences of M150 with LAMP-1. Sequence data of N-terminal and internal amino acid sequences of M150 in comparison with LAMP-1. Peptide sequence 1 was derived from the amino-terminal end of M150. Peptide sequences 2 and 3 are two internal sequences derived by tryptic digestion of M150 using a different preparation as reported by us earlier (23 ). D, Heteroduplex analysis. Heteroduplex analysis was conducted as described in Materials and Methods. The 500-bp PCR product generated from LAMP-1 cDNA was mixed with PCR product obtained from different clones (representing the RT-PCR product of thioglycolate-elicited macrophages). Duplex DNA thus generated was electrophoresed on 8% native PAGE and visualized by ethidium bromide staining. Lanes 1–3, Results for 3 of the 40 clones tested are shown as representatives.

 
To further confirm this, and to check whether there is any alternate form of LAMP-1 gene existing in macrophages with internal sequence differences as compared with 3T3 LAMP-1 cDNA, a heteroduplex analysis was performed. For this, corresponding oligonucleotide segments from the internal amino acid sequences of M150 were used as primers to amplify the corresponding segment from cDNA of LAMP-1. In parallel, these primers were also used to amplify the cDNA, isolated from thioglycolate-elicited macrophages. The amplified product was then cloned into an appropriate vector and used for transformation. The plasmid DNA from these colonies was then PCR amplified and subjected to a heteroduplex analysis with the PCR product derived from cDNA of LAMP-1. It may be mentioned in this study that mutations or sequence variations are easily detected by gel electrophoresis of heteroduplexes, where minute sequence variations appear as blebs on the gel. In contrast, identical sequences occur as homoduplexes. All of the 40 clones tested in hetroduplex analysis exhibited homoduplex formation. Results of three of these clones are shown in Fig. 1GoD as a representative example. Therefore, these results strongly suggest that M150 protein is in fact LAMP-1 protein, which is an activation-dependent cell surface glycoprotein also known as CD107a (34).

Differential expression of anti-M150 and anti-LAMP-1 reactivity in splenocyte subsets

The above results support that M150 and LAMP-1 are products of the same gene. Therefore, it is surprising that while LAMP-1 is a ubiquitous protein expressed in a variety of cell types, the expression of M150 is restricted to the surface of activated macrophages. To probe further for a possible basis for this distinction, we next examined the expression of M150 and anti-LAMP-1 reactivity in splenocyte subsets using specific Ab conjugates by FACS analysis. As shown in Fig. 2GoA, surface reactivity with anti-LAMP-1 Ab was displayed by activated splenic B cells, macrophages, and CD8+ T cells. In contrast, anti-M150 reactivity was restricted only to the Mac-1+ subset of activated splenocytes. This suggests that M150 is antigenically distinct from LAMP-1. However, as depicted in Fig. 2GoA, the anti-M150 and anti-LAMP-1 Abs have been found to bind only with the IFN-{gamma}-activated macrophage populations (i.e., the Mac-1+ populations of the splenocytes). Therefore, we tested the possibility of these two Abs for competing for the same epitope of LAMP-1 present on macrophage membrane. To address this issue, IFN-{gamma}-activated peritoneal exudate macrophages were stained with anti-M150 FITC-labeled Ab keeping the unlabeled anti-LAMP-1 Ab as a competitor of its specific site and vice versa. Fig. 2GoB demonstrates that there is no significant difference in the binding of either of the labeled Abs in the presence of the other as competitor, which conclusively proves that anti-LAMP-1 and anti-M150 Abs do not compete for binding to IFN-{gamma}-activated macrophages. Thus, this result also clearly substantiates that anti-LAMP-1 and anti-M150 bind to different epitopes of LAMP-1. However, NK T cells, dendritic cells, and monocytes were negative for staining with anti-LAMP-1 and anti-M150 Abs (data not shown).



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  		FIGURE 2. Differential surface expression of LAMP-1 and M150 on APCs and T cells. A, Flow cytometric analysis showing surface expression of LAMP-1 and M150 on splenocytes. Either resting (columns 1 and 2) or IFN-{gamma} activated (columns 3 and 4) splenocytes were analyzed for surface expression of LAMP-1 and M150. FITC-labeled anti-LAMP-1 and anti-M150 Abs were used along with PE-labeled Abs to the markers of B cells (B220), macrophages (mac-1), and T cells (CD4 and CD8) to double stain the splenocytes. PE-conjugated isotype controls of Abs were used for staining B cells (rat IgG2a), macrophages (rat IgG2b), CD4+ (rat IgG2b), and CD8+ T cells (rat IgG2a). Isotype controls for anti-LAMP-1 and anti-M150 Abs used were FITC-labeled normal rat IgG2a and normal rat IgM, respectively. a, The representative data of isotype controls used for staining splenocytes. b, Results for dual staining with anti-B220 PE and either FITC-conjugated anti-LAMP-1 or anti-M150 Abs. c, Dual staining was also performed using anti-Mac-1 PE along with either FITC-conjugated anti-LAMP-1 or FITC-labeled anti-M150 Abs. d, Staining of anti-CD4 PE- and FITC-labeled anti-LAMP-1 or anti-M150 Abs on splenocytes. e, The binding of anti-LAMP-1 FITC and anti-M150 FITC to CD8+ splenocytes. The experiments were done using the identical compensation throughout. In all of these experiments, >97% of the cells were viable as revealed by the propidium iodide staining (data not shown). B, Anti-LAMP-1 and anti-M150 recognize distinct epitopes on macrophages. Upper panel, The cells (106 cells/sample) were incubated simultaneously with 1.5 µg of anti-LAMP-1-FITC in the presence of purified anti-M150 at 5, 10, 25, and 50 times molar concentrations. Conversely, when cells were stained with anti-M150 FITC (as depicted in lower panel) 5, 10, 25, and 50 times molar concentrations of unlabeled anti-LAMP-1 was used as the competitor for the FITC-tagged anti-M150. Isotype controls for the anti-LAMP-1-FITC and anti-M150-FITC were FITC-conjugated normal rat IgG2a and normal rat IgM, respectively.

 
Differential glycosylation gives antigenic distinctness to LAMP-1

The existence of antigenic variance between M150 and LAMP-1 was also confirmed by Western blot analysis of the membrane fraction of activated macrophages using either anti-M150 or anti-LAMP-1 Abs as probes. Probing with either polyclonal or monoclonal anti-M150 Ab (G1) detected a band that centered around a molecular mass of 150 kDa. As opposed to this, anti-LAMP-1 Ab identified a lower band ranging from 120–145 kDa (Fig. 3GoA). Previous reports have demonstrated that the size heterogeneity of LAMP-1 is due to heterogeneous glycosylation (32). These studies also revealed that the size distribution of LAMP-1 differs significantly, depending upon the cell type examined. In other words, the glycosylation pattern of LAMP-1 appears to vary depending upon the cell type in which it is expressed (35).



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  		FIGURE 3. Western blot analysis showing differential glycosylation pattern of LAMP-1 in macrophages. A, Western blot analysis of macrophage membrane fractions. Surface membrane proteins from peritoneal exudate macrophages were isolated as described in Materials and Methods. These were then resolved by 10% SDS-PAGE and were transferred onto a nitrocellulose membrane and probed with different primary Abs. This was followed by incubation with biotinylated anti-rat and streptavidin-HRP. Bound secondary Ab was revealed using diaminobenzidine as the substrate. Lanes 1, 4, and 7, Protein molecular mass markers. Lanes 2 and 3, Position of M150 when the blots were probed with polyclonal anti-M150 and control rat sera, respectively. Lanes 5 and 6, The blots probed with anti-LAMP-1 and an isotype control rat IgG2a, respectively. Lanes 8 and 9, Position of M150 on the blots probed with monoclonal anti-M150 and isotype control rat IgM Abs, respectively. B, Western blot analysis of purified LAMP-1-Fc fusion proteins. Purified LAMP-1-Fc proteins (LAMP-1 fused with Fc), obtained from transfected CHO and P388D1 cells, were run on SDS-PAGE and transferred to a nitrocellulose membrane. Lanes 1 and 4, Biotinylated protein molecular mass markers. Lanes 2 and 5 contain CHO-LAMP-1-Fc, and lanes 3 and 6 contain macrophage-LAMP-1-Fc proteins. Left panel (lanes 2 and 3) was probed with anti-LAMP-1 Ab (ID4B) and right panel (lanes 5 and 6) was probed with anti-M150 monoclonal (G1) Ab. C, Deglycosylation of purified LAMP-1-Fc fusion proteins. Lane 1, Molecular mass markers; lanes 2 and 3, Deglycosylated products of CHO-LAMP-1-Fc and macrophage-LAMP-1-Fc proteins, respectively, separated on 10% SDS-PAGE. Note the identical mobility of both the proteins. D, lanes 1 and 2, Deglycosylated CHO-LAMP-1-Fc and macrophage-LAMP-1-Fc products (shown in C) probed with anti-Fc Ab.

 
To further confirm the possible cell type-specific differences in glycosylation of LAMP-1, we expressed a truncated version of LAMP-1 protein as LAMP-1-Fc chimera either in CHO cells, or in the mouse macrophage cell line, P388D1. The transfected fusion proteins were then purified and subjected to a Western blot analysis. As shown in Fig. 3GoB, nonidentical forms of the chimeric protein were produced in CHO and P388D1 cells, at least as detected by anti-LAMP-1 and anti-M150 mAbs. The product of CHO cells displayed a molecular mass between 105 and 120 kDa; in contrast, the product from P388D1 cells was significantly of higher molecular mass, ranging from 130–150 kDa. Interestingly, anti-M150 mAb did not show reactivity with LAMP-1 protein expressed in CHO cells while recognizing the LAMP-1 expressed in macrophages (Fig. 3GoB, compare lanes 5 and 6). In contrast, anti-LAMP-1 recognized a discrete number of lower molecular mass proteins from LAMP-1-transfected CHO and P388D1 cells as shown in Fig. 3GoB, lanes 2 and 3, respectively. A comparison of the protein bands (Fig. 3GoB, lanes 3 and 6) recognized by anti-LAMP-1 and anti-M150 Abs suggest that the M150 is a specific form which constitutes only a small fraction of the total LAMP-1 produced in macrophages.

To compare the protein core of these two molecular species, the expressed products from both CHO and P388D1 cells were first deglycosylated using endoglycosidase H. Surprisingly, this procedure completely eliminated the reactivity of the anti-LAMP-1 and anti-M150 Abs. Thus, the resultant deglycosylated proteins could be detected only with anti-Fc Ab.

Therefore, these results collectively suggest that both Abs preparations are directed against the glycosylated portion of the molecules. Thus, from these results it can also be inferred that the expression of LAMP-1-Fc chimera in CHO and P388D1 cells results from qualitatively distinct glycosylation patterns. Furthermore, it is this variation in glycosylation that accounts for the observed difference in antigenicity, at least with respect to anti-M150 Ab. In other words, the anti-M150 mAb appears to be directed against a LAMP-1 subset that is specifically produced only in macrophages.

The LAMP-1-Fc chimera displays costimulatory activity when expressed in macrophages

We next examined whether the altered glycosylation pattern of the LAMP-1-Fc chimera also confers altered function for the protein molecule when expressed in P388D1 vs CHO cells. Therefore, purified CD4+ T cells were stimulated with varying concentration of anti-CD3 Ab in the presence of a fixed concentration of either CHO-LAMP-1-Fc or macrophage-LAMP-1-Fc proteins. A proliferative response was observed only in cultures that included macrophage-LAMP-1-Fc, but not in those where CHO-LAMP-1-Fc was added (Fig. 4GoA).



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  		FIGURE 4. CD4+ T cell proliferation assay and cytokine analysis in response to LAMP-1-Fc. A, Induction of CD4+ T cell proliferation induced by macrophage-LAMP-1-Fc. T cell proliferation assay was performed in the presence of increasing concentrations of soluble anti-CD3 Ab, without ({square}) or with () Fc control, or with CHO-LAMP-1-Fc () and macrophage-LAMP-1-Fc, i.e., M{phi}-LAMP-1-Fc ({blacksquare}), used at a concentration of 2 µg/ml. Proliferation was measured by [3H]thymidine incorporation during the last 16 h of the 72-h culture. The data represent the mean of three independent experiments and SD was obtained by nonbiased or (n-1) method. B, Anti-M150 blocks the T cell proliferation induced by macrophage LAMP-1-Fc. The T cell proliferation assay was performed (as described for A) in presence of soluble anti-CD3 Ab (1 µg/ml) and Fc protein ({square}), CHO-LAMP-1-Fc (), and M{phi}-LAMP-1-Fc ({blacksquare}) at the indicated concentrations. To describe the effect of various Abs in the blocking assay, the bars are labeled as a, b, c, and d, which represent anti-M150, normal rat IgG2a, anti-LAMP-1, and normal rat IgM, respectively. The proliferation induced by macrophage LAMP1-Fc at 0.5 µg/ml concentration in the presence of anti-CD3, was inhibited by only anti-M150 Ab (a), where as its isotype control, rat IgM (d) was found to have no effect. No effect of anti-LAMP-1 Ab (c) or its isotype control rat IgG2a (b) on the T cell proliferation was noticed. The anti-M150 Ab blocked the proliferation to a significant level induced by macrophage-LAMP-1-Fc. The data represent the mean of three independent experiments and SD was obtained by nonbiased or (n-1) method. C, Induction of Th1 type cytokines by macrophage-LAMP-1-Fc. Culture supernatants from the T cell proliferation assay with increasing concentrations of purified macrophage LAMP1-Fc in the presence of anti-CD3 (1 µg/ml) show the presence of IL-2 ({blacksquare}) and IFN-{gamma} ({square}). There were no detectable levels of these cytokines when CHO-LAMP-1-Fc was used to costimulate the Th cells. IL-4 and IL-13 were not detectable in these supernatants. The data represent the mean of three independent experiments and SD was obtained by nonbiased or (n-1) method.

 
It is possible that the difference in activity observed for macrophage-LAMP-1-Fc and CHO-LAMP-1-Fc in Fig. 4GoA simply reflects differences in dose requirements for the two chimeric products. To verify this, T cells were stimulated with a constant dose of 1 µg/ml of anti-CD3 Ab in the presence of increasing concentrations of either macrophage-LAMP-1-Fc or CHO-LAMP-1-Fc. For these experiments, a dose-dependent proliferation of CD4+ T cells was obtained with macrophage-LAMP-1-Fc (Fig. 4GoB). In contrast to this, CHO-LAMP-1-Fc was unable to induce T cell proliferation at any of its tested concentrations (Fig. 4GoB). Furthermore, the specificity of the macrophage-LAMP-1-Fc-dependent T cell response is also evident from the fact that this effect could be inhibited by addition of anti-M150 Ab, but not by anti-LAMP-1 Ab (Fig. 4GoB).

We have previously demonstrated that M150 serves as a costimulatory molecule that specifically elicits cytokines typical of the Th1 subset of CD4+ T cells. As shown in Fig. 4GoC, the macrophage-LAMP-1-Fc product also reproduced this activity when used in conjunction with anti-CD3 for stimulation of CD4+ T cells. Significant levels of both IL-2 and IFN-{gamma} were detected in the supernatants from these cultures (Fig. 4GoC). In contrast, stimulation in presence of CHO-LAMP-1-Fc failed to induce detectable levels of Th1 representative cytokines (Fig. 4GoC). It is pertinent to mention that no detectable levels of Th2 representative cytokines were obtained from the culture supernatants of Th cells activated either by CHO-LAMP-1-Fc or macrophage LAMP-1-Fc. Collectively, these results categorically identify that LAMP-1 can indeed display Th1-specific costimulatory activity, but this activity is contingent upon its expression in macrophages.

Macrophage-LAMP-1-Fc induces expression of T-bet in naive CD4+ T cells

It is now becoming evident that differentiation of CD4+ T cells into either the Th1 or Th2 commitment pathways is regulated by the activation of independent transcription factors (36). Subset-specific transcription factors have been identified and their role in distinct T cell differentiation is being actively explored. For example, STAT-6 has been shown to synergize with an antigenic stimulus leading to the up-regulation of GATA3, a potent inducer of Th2 differentiation (31). The transcription factor c-Maf, which negatively regulates Th1 differentiation, has now been shown to be responsible for the tissue-specific expression of IL-4 (37). Recently, T-bet was identified as a transcription factor specific to Th1 cells, and demonstrated to act by controlling the expression of IFN-{gamma} (38). Given the Th1-specific costimulatory activity observed for M150 in our earlier study (23), as well as in the present report (Fig. 4GoC), we further examined whether it could be implicated in selectively driving differentiation of CD4+ T cells.

CD4+ T cells were stimulated with anti-CD3 in the presence of either macrophage-LAMP-1-Fc or CHO-LAMP-1-Fc from which the total RNA was isolated. This was then analyzed for the presence of specific mRNA for c-Maf and T-bet by RT-PCR. Consistent with the absence of any activity of CHO-LAMP-1-Fc, no expression of either c-Maf or T-bet could be detected in cells stimulated with this protein. In contrast, costimulation with macrophage-LAMP-1-Fc yielded a significant induction of T-bet mRNA, with no concomitant effect on c-Maf expression (Fig. 5Go). Therefore, this selective effect of macrophage-LAMP-1-Fc on T-bet expression may implicate M150, a modified glycosylated form of LAMP-1, as a costimulatory molecule that specifically drives the naive CD4+ T cells toward Th1 differentiation.



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  		FIGURE 5. Induction of T-bet, a Th1-specific transcription factor. Total RNA was prepared from CD4+ T cells activated in presence of anti-CD3 (1 µg/ml) and with CHO-LAMP-1-Fc (2 µg/ml), or with macrophage-LAMP-1-Fc for 24 h and analyzed for the messages for c-Maf and T-bet by RT-PCR. Lanes 1 and 2, Equal loading controls which are the RT-PCR product for dihydrofolatereductase (DHFR) from the RNA prepared from T cells activated with CHO-LAMP-1-Fc protein and M{phi}-LAMP-1-Fc fusion proteins, respectively. Lanes 3 and 4, The RT-PCR products of c-Maf and T-bet mRNA, respectively, from T cells activated with CHO-LAMP-1-Fc. Lane 5 and 6, The RT-PCR product of c-Maf and T-bet mRNA derived from M{phi}-LAMP-1-Fc-activated T cells, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The development of a productive T cell response initially depends upon appropriate costimulatory signals that are provided by the APC. Costimulatory molecules present on the surface of activated APCs are now known to act by binding to specific receptors on T cells which, in turn, initiate intracellular signaling pathways that lower the threshold for T cell activation. Although it was earlier thought that CD80 and CD86 were the principal costimulatory molecules involved in regulation of immune responses, more recent studies have revealed that they constitute members of a family of related molecules. As discussed earlier, several novel costimulatory molecules have now been identified, some of which display selectivity either at the level of APC-restricted expression or at the level of Th subset preferences. Thus, immune regulation appears to be far more complex than hitherto suspected, the outcome being presumably dependent on a tightly controlled network of interactions between the various costimulatory molecules and their appropriate receptor on T cells. Therefore, T cell activity may well represent a temporally regulated process, which at any given time will be dependent on both the spectrum and surface densities of individual costimulatory molecules that are expressed on the APC surface.

In a recent study, we had isolated a novel protein M150 from the plasma membrane of activated macrophages that possessed costimulatory activity (23). We also demonstrated further that the activity of M150 dominates over that of B7.1 when both are expressed on the surface of activated macrophages. This was demonstrated both at the level of enhanced alloreactivity, as well as increased IFN-{gamma} production in cocultures with T cells (24).

The present report is the outcome of our subsequent efforts to further characterize the M150 protein. Surprisingly, our results revealed that M150 was in fact the ubiquitously expressed LAMP-1, albeit in a uniquely glycosylated form. This was initially suggested from a partial amino acid sequence analysis of the intact M150 protein. Our inference was further supported by heteroduplex analysis, which unambiguously demonstrated the identity between M150 and LAMP-1. Finally, expression of the extracellular domain part of LAMP-1 as a fusion protein in macrophages, but not CHO cells, also yielded a product with M150-like activity.

The functional properties of LAMP-1 continue to remain elusive. Observations that LAMP-1 expression on the surface of several tumors increases their metastatic potential suggest a role for this protein in mediating cell adhesion to vascular endothelium (39). However, direct evidence that supports either an in vivo function, or a physiological role for LAMP-1 is currently lacking. A particularly striking aspect of our results is the finding that a uniquely glycosylated form of LAMP-1 possesses costimulatory activity. To our knowledge, this is the first report, which indicates that T cell costimulating potential can also derive from posttranslational modification of an otherwise inactive molecule. However, the nature of glycosylation that confers costimulatory activity to LAMP-1 remains to be characterized. Our present results particularly underscore the versatility that is built into immune regulatory processes. Thus, in addition to using "professional" costimulatory molecules, recruitment of housekeeping proteins by selective posttranslational modifications may constitute an additional pathway by which T cell responses may be modulated. A possible significance of the latter pathway could be that it facilitates APC-specific effects on T cell costimulation. Significantly, our results demonstrate that costimulation of T cells with macrophage-LAMP-1-Fc induces expression of T-bet may also implicate a role of M150 in driving differentiation of naive CD4+ T cells into the Th1 pathway.

In summary, M150 is a uniquely glycosylated form of LAMP-1 that is produced only in activated macrophages. However, it remains to be ascertained if LAMP-1 represents an isolated example, or if there are other such housekeeping molecules that can also function as costimulatory molecules under specific circumstances. As the catalogue of APC-surface molecules that provide costimulation to T cells increases, the question of their cumulative involvement in the regulation of immune responses also assumes increasing importance. It would also be to determine whether this apparent redundancy of costimulatory molecules simply provides a safeguard against evasive strategies evolved by pathogens, or if they constitute a cooperative network that appropriately regulates the response depending on the nature of "danger" perceived (40).


   Acknowledgments
 
We thank Drs. Tak W. Mak and S. K. Yoshinaga SK for providing pCEP4-Fc vector, and Dr. J. Thomas August for providing LAMP-1 cDNA. We are extremely grateful to Dr. K. V. S. Rao (International Center for Genetic Engineering and Biotechnology, New Delhi, India) for critical evaluation and suggestions. A part of the manuscript was presented to National Academy of Sciences (Allahabad, India) as presidential address (Biological Section). We also thank Drs. A. Sahu, K. Shastry, and S. Bapat for critical reading of the manuscript, and Atul Suple for excellent technical assistance.


   Footnotes
 
1 This work was supported by Department of Biotechnology, Government of India. D.V.R.P. and P.P.B. are the recipients of Senior Research Fellowship from National Centre for Cell Science, Department of Biotechnology, Pune, India. V.V.P. is a recipient of Senior Research Fellowship, and B.N.J. is a recipient of Research Associateship from Council for Scientific and Industrial Research, New Delhi, India. Back

2 Address correspondence and reprint requests to Dr. Gyan C. Mishra, National Center for Cell Science, Ganeshkhind Road, Pune 411007, Maharashtra, India. E-mail address: gcmishra{at}nccs.res.in Back

3 Abbreviations used in this paper: LAMP-1, lysosome-associated membrane protein-1; CHO, Chinese hamster ovary. Back

Received for publication October 23, 2001. Accepted for publication June 17, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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