![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1
2To whom correspondence should be addressed. E-mail: wwoodwar{at}uoguelph.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
2% of initial body weight daily. Naïve-phenotype (quiescent) CD8+ T cells of the blood, spleen and mesenteric lymph nodes were identified on the basis of surface coexpression of CD45RA and CD62L. Relative to age-matched controls, the percentage of naïve-phenotype CD8+ T cells was high in energy-restricted groups after 9 d and 14 d of weight loss and in the protein-restricted groups after 14 d (P 
0.05). No ontogenetic change was apparent (age-matched vs. zero-time control). Other studies have demonstrated depression in cell-mediated immune competence in both malnutrition models within the first week of weight loss. An overabundance of quiescent-phenotype T cells within the involuted CD8+ compartment may contribute to established immune depression but not to its initiation in weight loss pathologies.
KEY WORDS: • mice • T cell • blood • spleen • lymph node
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). However, PEM-associated depression in T-dependent immune competence can be prevented (2
–4
) or even reversed (5
), despite unabated and profound lymphoid atrophy in which cellular losses exceed 90%. It is reasonable, therefore, to pursue other features of the immunobiology of PEM, whether deficiency of protein or energy, which may prove more basic than lymphoid involution to depressed acquired immunity in wasting disease.
Imbalances among subsets of T cells may contribute to PEM-associated immunodepression (1
). This proposition has received support recently from the study of wasting protein and energy deficiencies in the weanling mouse. During the advanced stages of these metabolically dissimilar pathologies, an overabundance of cells expressing CD45RA arises among CD4+ T cells throughout the involuted lymphoid system (6
,7
). The CD4+CD45RA+ (naïve-phenotype) T cell is quiescent relative to its CD45RA- (memory/effector) counterpart in terms of proliferative and cytokine response capacity (8
,9
). Thus, an imbalance favoring quiescence within the CD4+ T cell subset may contribute to depressed T-dependent immunocompetence in the advanced stages of weanling protein or energy deficit (6
,7
).
A similar shift toward overabundance of CD45RA+ cells occurs within the CD8+ T cell subset in the wasted, protein- or energy-deficient weanling mouse (6
). However, expression of CD45RA is insufficient to distinguish naïve-type cells within this subset because a substantial proportion of CD45RA+CD8+ T cells exhibit the functional characteristics of effector cells (9
). Thus, it is unclear whether an imbalance favoring a quiescent phenotype occurs among CD8+ T cells in acute weight loss pathologies, or whether this phenomenon is confined to the CD4+ T cell subset. This is important to determine, however, because the CD8+ subset includes both cytotoxic effector cells (10
) and cytokine-producing regulatory cells (11
). Naïve-type CD8+ T cells can be identified as CD45RA+CD62L+ elements that exhibit quiescence relative to CD45RA-CD62L- (effector- and memory-phenotype) cells in terms of proliferative and cytokine-producing activity in response to stimulation through the T cell receptor (9
,12
,13
). The objective of the present investigation, therefore, was to determine whether a shift toward a quiescent phenotype occurs among CD8+ T cells in metabolically distinct models of acute weanling malnutrition known to produce depression in acquired immune competence and, if so, at what stage this occurs in the progression of weight loss pathology.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Male and female C57BL/6J mice were used from an in-house breeding colony. Caging and housing conditions were exactly as described previously (4
,6
,7
,14
,15
), and the investigation was approved by the Animal Care Committee of the University of Guelph in accordance with the Canadian Council on Animal Care.
Study design, diets and feeding protocols.
Mice were weaned at 18 d of age, acclimated for 1 d to a complete purified diet and, subsequently, allocated to experimental groups. These included two malnourished groups, an age-matched control group and a zero-time control, the latter (19 d of age) to control for ontogeny-related change. The age-matched control group was given free access to a complete purified diet described elsewhere (14
,15
). This diet meets or exceeds current standards of the National Research Council (16
) for laboratory mice, and a typical proximate analysis is 92.3%, dry matter; 18.8%, crude protein; 8.1%, ether extract; 2.6%, ash; 3.1%, crude fiber; and 17.0 kJ/g, gross energy (4
). One group of malnourished mice was fed restricted quantities of the complete diet each day. The quantity of diet provided to each mouse of this group was determined as described previously (7
) according to a procedure yielding a high degree of uniformity among mice in achieving a loss of 2% of initial body weight per day. A second group of malnourished mice was given free access to a low-protein diet. This diet, described elsewhere (14
), was isocaloric with the complete diet but contained 0.6% crude protein. All mice had constant unimpeded access to clean tap water, and coprophagy was permitted as in previous studies of T cells and immune competence in the same experimental systems (2
–7
,14
,15
).
Three separate feeding trials were conducted. The results of the first study, which was 14 d in duration, gave rise to the second experiment with a 9-d feeding period. The third experiment was a 6-d trial and did not include mice fed the low-protein diet. Sample sizes of eight mice (four males and four females) were achieved for each group in each experiment except that 7 mice were included in the 9-d age-matched control group and in the 14-d zero-time control group. Because of low cell numbers, pooling was necessary for all malnourished groups (two mice per sample). Each pooled sample comprised one gender and constituted a single degree of freedom for the purpose of statistical analysis.
Procedures to obtain blood samples, spleen and lymph nodes.
Procedures to obtain blood, spleen and nodal tissue have been described (6
,7
,14
). Briefly, blood samples from the orbital plexus of mice anesthetized with diethyl ether (Fisher Scientific, Fair Lawn, NJ) were collected into heparinized microcentrifuge tubes. Mice were then killed by cervical dislocation, and the spleen and mesenteric nodes were removed aseptically into RPMI 1640 medium (Flow Laboratories, Mississauga, Canada) containing 10% heat-inactivated fetal calf serum (Sigma, St. Louis, MO) and 1 mmol/L HEPES (ICN Biomedicals Canada, St. Laurent, Canada).
Identification of cellular subsets by flow cytometry.
Single-cell suspensions of mononuclear cells were prepared from the spleen, mesenteric lymph nodes and blood as described (6
,7
,14
). Viability before staining was determined by eosin Y exclusion and always exceeded 95%. Cellular subsets were identified using a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Mississauga, Canada) equipped with System II software, version 3 (1998). Cells were subjected to Fc receptor blockade after which a three-stage staining procedure was followed. A description of staining procedures as performed in this laboratory, including Fc receptor blockade, is provided elsewhere (6
,14
,15
). Briefly, the cells were first stained with biotin-conjugated anti-CD8
(YTS 169.4, rat IgG2b; Cedarlane Laboratories, Hornby, Canada) together with unconjugated anti-CD45RA (RA3–2C2/1, rat IgM), the latter reagent having been produced and purified in this laboratory as described elsewhere (15
). Second, the cells were stained with phycoerythrin (PE)-indodicarbocyanine (Cy5)-conjugated streptavidin (Cedarlane Laboratories) to reveal CD8+ cells and were stained also with fluorescein isothiocyanate (FITC)-conjugated IgG F(ab)2 fragment of goat anti-rat µ-heavy chain (Bio/Can Scientific, Mississauga, Canada) to reveal CD45RA+ cells. Finally, the cells were stained with PE-conjugated anti-CD62L (MEL-14, rat IgG2a). Negative control samples were produced by a three-stage staining procedure in which biotin-conjugated rat IgG2b (Cedarlane Laboratories) was followed by PE-Cy5-conjugated streptavidin together with FITC-conjugated IgG F(ab)2 fragment of goat anti-rat µ-heavy chain and, finally, with PE-conjugated rat IgG2a (Cedarlane Laboratories). Each analysis was based on at least 104 events after dead cells and residual erythrocytes were eliminated by gating on the basis of forward-angle light scatter. In this analysis, both CD8+ staining intensity and forward-angle light scatter aided distinction of CD8+ T lymphocytes from other cells, e.g., macrophages, which express the CD8 surface marker.
Carcass analyses.
Carcasses were stored at -20°C for not >18 wk before analysis. Dry matter, lipid and crude protein contents were determined as described (4
,14
,15
).
Statistical analysis.
The predetermined upper limit of probability for statistical significance throughout this investigation was P 0.05. Data were subjected to two-tailed Student’s t test or to one-way ANOVA followed (if justified by the resulting statistical probability value, i.e., P 0.05) by either Duncan’s New Multiple Range test or Dunnett’s t test. If the error term of a data set failed to exhibit normal distribution after application of several transformation procedures, the Kruskal-Wallis test (
2 approximation) was applied to Wilcoxon rank sums and, where warranted by the resulting statistical probability (i.e., P 0.05), this analysis was followed by 2 comparisons of Wilcoxon two-sample rank sums for each combination of treatment pairs. Where data sets were transformed to normality, transformed means and pooled SEM values are presented.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Growth indices are shown in Table 1
for each of the three experiments (feeding periods) conducted. Initial body weights did not differ among groups in any of the three experiments. The age-matched control groups exhibited food intakes and gains of fat and lean tissue that were comparable to outcomes reported previously in studies of C57BL/6J weanlings given free access to the complete diet (7
). The two malnutrition protocols induced low food intakes and comparable weight loss. However, mice subjected to food intake restriction exhibited a greater loss of carcass lipid than did corresponding groups fed the low-protein diet. Overall, therefore, the wasting disease produced by each type of malnutrition protocol was comparable to that reported in the same experimental systems in studies demonstrating early depression in T-dependent immunocompetence (4
,7
) and a later shift toward quiescent, naïve-type phenotype among CD4+ T cells (6
,7
,15
). Mononuclear and CD8+ T cell counts also were consistent with this conclusion. As is appropriate to these experimental systems (4
,6
,7
,14
,15
), ontogenetic increase was apparent both in total mononuclear cell numbers and in CD8+ T cell numbers within each lymphoid compartment examined in the age-matched control groups (compared with zero-time control), whereas involution of these cell populations occurred at each stage examined in both forms of weight loss pathology (results not shown).
|
The percentages of CD8+ T cells expressing both CD45RA and CD62L within the mononuclear cell suspensions of the blood, spleen and mesenteric lymph nodes are shown in Figure 1
. After 14 d of weight loss, both malnourished groups exhibited high proportions of quiescent-phenotype cells within the CD8+ subset in all three lymphoid compartments relative to the age-matched control group. In contrast, only the energy-restricted mice exhibited this imbalance at 9 d of weight loss. Because of this outcome, an experiment was conducted in which mice were subjected to only 6 d of food intake restriction, but no diet-related influence was apparent on the proportion of CD8+ T cells expressing a naïve-type surface phenotype at this stage of weight loss. Comparison of age-matched and zero-time control groups revealed no ontogeny-related change in this index.
|
. Comparison of age-matched and zero-time control groups revealed an ontogenetic increase in cell numbers within this subset by 33 d of age in all three lymphoid compartments. In contrast, involution of the naïve-phenotype subset (i.e., compared with the zero-time control) was apparent in the spleen and mesenteric nodes by the earliest stage of weight loss at which each form of malnutrition was examined. No clear effect of either malnutrition protocol was apparent on blood numbers of naïve-phenotype CD8+ T cells (µL-1) compared with zero-time controls, but both protocols produced low numbers of naïve-phenotype blood cells (µL-1) relative to age-matched controls at all stages of weight loss examined.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
,12
,13
). This subset includes a small population of effector cells (9
) but, nevertheless, exhibits quiescence in terms of proliferative and cytokine-producing capacity (9
,12
,13
). The present investigation reveals a relative overabundance of such quiescent-phenotype CD8+ T cells throughout the involuted peripheral lymphoid system in the more advanced stages of metabolically diverse forms of weight loss pathology. Previous studies of the malnutrition protocols used in this investigation show depressed rejection of complete major histocompatibility disparate skin allografts, a CD8+ T cell-dependent response (17
), within the first week of weight loss (4
), i.e., before the appearance of the subset imbalance identified herein. Nevertheless, this imbalance phenomenon may contribute to ongoing immune depression in the advanced stages of wasting pathology.
Several features of the experimental design used in the present study promote confidence in the broad applicability of the results. First, the spleen and mesenteric lymph nodes are the largest encapsulated secondary lymphoid organs in the mammalian body (18
), and the mesenteric nodes include lymphocytes from both the systemic and the mucosal systems (19
). Second, both male and female mice were included so as to reveal any gender-specific influences of PEM during the postweaning period extending to adolescence. Third, inclusion of a zero-time control group revealed that no ontogeny-related change occurred in the proportion of CD8+ T cells coexpressing CD45RA and CD62L during the stage of life examined in the present study. Thus, the PEM-associated subset imbalance identified herein reflects a more fundamental phenomenon than simply an influence on immunological ontogeny. Finally, the study was conducted using metabolically dissimilar malnutrition protocols, one relevant to marasmus and the other to incipient kwashiorkor (2
,14
). In this connection, the restricted intake protocol produces weight loss as a result of energy deficiency while protein and micronutrients are in sufficient supply (14
), whereas the low-protein protocol produces wasting as a result of nitrogen deficiency in the presence of sufficient intake of energy and micronutrients (6
).
The shift toward a naïve CD8+ T cell phenotype occurred earlier in mice subjected to energy restriction than in mice subjected to protein deficiency, and the same outcome has been reported in relation to the CD4+ subset (6
,7
). As in previous studies (7
,14
,15
), carcass lipid analyses demonstrated that the low-intake protocol imposed a greater rate of energy deficit than the protein deficiency protocol. Thus, negative energy balance may be a determining factor in the wasting-associated imbalance toward naïve phenotype among T cells of both major subsets, regardless of the metabolic form of weight loss pathology.
Some differences are noteworthy between the PEM-associated naïve-phenotype imbalances in the CD4+ (6
,7
) and the CD8+ (this investigation) T cell subsets. In particular, the quiescent-phenotype population of CD4+ T cells resists involution even into advanced weight loss (7
), whereas the corresponding population of CD8+ T cells failed to exhibit such remarkable stability. Moreover, comparison with reports pertaining to CD4+ T cells (6
,7
) reveals that the shift toward a quiescent phenotype occurs first within the CD8+ subset. The basis for such subset-specificity is not known. However, effector- and memory-phenotype T cell populations, even resting memory cells, exhibit rapid turnover relative to populations of naïve-phenotype T cells (20
). This seems unimportant to the imbalance phenomenon within the CD4+ subset (7
), but it may be a dominant factor among CD8 + T cells.
Perhaps the main distinguishing feature of effector and memory T cells, both CD4+ and CD8+, is their capacity to produce a rich diversity of cytokines and, thereby, to fulfill a helper function (11
). Low T helper activity seems central to immune incompetence in PEM (1
), but the long-standing reliance on depression in the CD4/CD8 ratio as a basis for PEM-associated helper incapacity is no longer tenable (1
,14
). Emergence of an imbalance phenomenon that extends throughout the T cell system and that is potentially relevant to helper activity in metabolically diverse weight loss pathologies, therefore, is significant. The results of the present investigation complete the phenotypic evidence for a shift toward naïve-type quiescence throughout the T cell system in wasting disease and highlight the need for studies of function including cytokine production.
|
FOOTNOTES |
|---|
3 Abbreviations used: Cy5, indodicarbocyanine; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PEM, protein-energy malnutrition; T, thymus.
Manuscript received July 27, 2001. Initial review completed August 19, 2001. Revision accepted September 21, 2001.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Woodward, B. (1998) Protein, calories and immune defenses. Nutr. Rev. 56(suppl. 1):S84-S92.[Medline]
2. Filteau, S. M., Perry, K. J. & Woodward, B. (1987) Triiodothyronine improves the primary antibody response to sheep red blood cells in severely undernourished weanling mice. Proc. Soc. Exp. Biol. Med. 185:427-433.[Medline]
3. Perry, K. J., Filteau, S. M. & Woodward, B. (1988) Dissociation of immune capacity from nutritional status by triiodothyronine supplements in severe protein deficiency. FASEB J 2:2609-2612.[Abstract]
4. Woods, J. W. & Woodward, B. D. (1991) Enhancement of primary systemic acquired immunity by exogenous triiodothyronine in wasted, protein-energy malnourished weanling mice. J. Nutr. 121:1425-1432.
5. Woods, J. W. & Woodward, B. (1994) Immunorestorative effect of triiodothyronine supplementation on the primary antibody response to sheep red blood cells following the development of immunodepression in protein-energy malnourished weanling mice. J. Nutr. Immunol. 3:3-12.
6. Woodward, B., Hillyer, L. & Hunt, K. (1999) T cells with a quiescent phenotype (CD45RA+) are overabundant in the blood and involuted lymphoid tissues in wasting protein and energy deficiencies. Immunology 96:246-253.[Medline]
7.
Ha, C. L., Wong, S.S.L., Gray, M. M., Watt, J., Hillyer, L. M. & Woodward, B. D. (2001) Overabundance of CD45RA+ (quiescent-phenotype) cells within the involuted CD4+ T-cell population follows initiation of immune depression in energy-deficient weanling mice and reflects involution exclusive to the CD45RA- subset. J. Nutr. 131:1812-1818.
8. Lightstone, E., Marvel, J. & Mitchison, A. (1993) Hyper-reactivity of mouse CD45RA- T cells. Eur. J. Immunol. 23:2383-2386.[Medline]
9. Sallusto, F., Lenig, D., Forster, R., Lipp, M. & Lanzavecchia, A. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712.[Medline]
10. Zajac, A. J., Murali-Krishna, K., Blattman, J. N. & Ahmed, R. (1998) Therapeutic vaccination against chronic viral infection: the importance of cooperation between CD4+ and CD8+ T cells. Curr. Opin. Immunol. 10:444-449.[Medline]
11. Dutton, R. W., Bradley, L. M. & Swain, S. L. (1998) T cell memory. Annu. Rev. Immunol. 16:201-223.[Medline]
12.
Hamaan, D., Baars, P. A., Rep, M.H.G., Hooibrink, B., Kerkhof-Garde, S. R., Klein, M. R. & van Lier, R.A.W. (1997) Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med. 186:1407-1418.
13.
Mitra, D. K., de Rosa, S. C., Luke, A., Balamurugan, A., Khaitan, B. K., Tung, J., Mehra, N. K., Terr, A. I., O’Garra, A., Herzenberg, L. A., Herzenberg, L. A. & Roederer, M. (1999) Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases. Int. Immunol. 11:1801-1810.
14. Lee, W. H. & Woodward, B. D. (1996) The CD4/CD8 ratio in the blood does not reflect the response of this index in secondary lymphoid organs of weanling mice in models of protein-energy malnutrition known to depress thymus-dependent immunity. J. Nutr. 126:849-859.
15. Woodward, B. D., Bezanson, K. D., Hillyer, L. M. & Lee, W. H. (1995) The CD45RA+ (quiescent) cellular phenotype is overabundant relative to the CD45RA- phenotype within the involuted splenic T cell population of weanling mice subjected to wasting protein-energy malnutrition. J. Nutr. 125:2471-2482.
16. National Research Council (1995) Nutrient requirements of the mouse. Nutrient Requirements of Laboratory Animals 4th rev. ed. 1995:80-102 National Academy Press Washington, DC. .
17. Auchincloss, H., Jr, Mayer, T., Ghobrial, R. & Winn, H. J. (1989) T-cell subsets, bm mutants, and the mechanisms of allogeneic skin graft rejection. Immunol. Res. 8:149-164.[Medline]
18. Hunt, S. V. (1987) Preparation of lymphocytes and accessory cells. Klaus, G.G.B. eds. Lymphocytes: A Practical Approach 1987:1-34 IRL Press Oxford, UK. .
19. Olszewski, W. L. (1987) The migration of the lymphoblast. In Vivo Migration of Immune Cells 1987:67-81 CRC Press Boca Raton, FL. .
20. Sprent, J. & Surh, C. D. (2001) Generation and maintenance of memory T cells. Curr. Opin. Immunol. 13:248-254.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. R. Foote, B. J. Nonnecke, D. C. Beitz, and W. R. Waters High Growth Rate Fails to Enhance Adaptive Immune Responses of Neonatal Calves and Is Associated with Reduced Lymphocyte Viability J Dairy Sci, January 1, 2007; 90(1): 404 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Monk, K. Makinen, B. Shrum, and B. Woodward A BRIEF COMMUNICATION: Blood Corticosterone Concentration Reaches Critical Illness Levels Early During Acute Malnutrition in the Weanling Mouse. Experimental Biology and Medicine, March 1, 2006; 231(3): 264 - 268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Foote, B. J. Nonnecke, M. A. Fowler, B. L. Miller, D. C. Beitz, and W. R. Waters Effects of Age and Nutrition on Expression of CD25, CD44, and L-Selectin (CD62L) on T-cells from Neonatal Calves J Dairy Sci, August 1, 2005; 88(8): 2718 - 2729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Nonnecke, M. R. Foote, J. M. Smith, B. A. Pesch, and M. E. Van Amburgh Composition and Functional Capacity of Blood Mononuclear Leukocyte Populations from Neonatal Calves on Standard and Intensified Milk Replacer Diets J Dairy Sci, November 1, 2003; 86(11): 3592 - 3604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhang, L. M. Hillyer, and B. D. Woodward The Capacity of Noninflammatory (Steady-State) Dendritic Cells to Present Antigen in the Primary Response Is Preserved in Acutely Protein- or Energy-Deficient Weanling Mice J. Nutr., September 1, 2002; 132(9): 2748 - 2756. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
