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Proceedings of the Society for Experimental Biology and Medicine 224:187-190 (2000)
© 2000 Society for Experimental Biology and Medicine

Original Article

Effects of Inositol, LiCl, and Heparin on the Antibody Responses to SRBC by Normal and Immunodeficient XID Mice

Marvin L. Tyan1,


West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Spleen cells from naïve adult immunocompetent and immunodeficient XID mice were cultured on agar containing sheep red blood cells (SRBC) with and without myo-inositol, scyllo-inositol, lithium chloride, or heparin, and after 1 or 2 days the number of colonies of antiSRBC antibody–forming cells (PFC) were determined. It was found that myo-inositol and scyllo-inositol at one-tenth the concentration were equally effective in increasing the number of specific PFC. Myo-inositol, scyllo-inositol, and lithium chloride accelerated the appearance of direct foci in cultures of spleen cells from normal and XID mice. When heparin was added to cultures of XID spleen cells, PFC were found to be increased on Day 1; however, PFC and foci were not increased in cultures of spleen cells from competent mice until 1 day later. The addition of combinations of these agents to cultures of spleen cells had no positive or negative effect on the generation of foci or PFC. Normal mice given heparin intraperitoneally with SRBC had increased splenic PFC on Days 3 and 4 but not on Day 7. The results suggest that these agents modulate B-cell responses by increasing the rate of proliferation and/or secretion through a signaling pathway(s) distal to, or more likely, independent of Bruton's tyrosine Kinase (BTK). It is not clear that the mechanism is the same with each agent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Previous reports have found (1) that the rate of appearance of antibody-forming cells (PFC) in the spleens of inbred mice is accelerated in a primary response to sheep red blood cells (SRBC) if the mice are fed a diet supplemented with myo-inositol (4 mg/g of diet). In vitro, myo-inositol accelerated the rate of appearance of colonies of antiSRBC PFC (foci) and significantly increased the number of PFC per colony but not the total number of colonies noted at the end of the culture period. Myo-inositol had no effect on the PFC IgM:IgG ratio following a single exposure to the agent in vitro, but exposure to myo-inositol both in vivo and in vitro resulted in a decrease in the number of IgM PFC per colony in a primary response and IgM and IgG PFC per focus in a secondary response.

When immunodeficient CBA/CaHN-XID/J mice that have a defect in Bruton's tyrosine kinase (2, 3) were fed the diet supplemented with myo-inositol, they produced significantly more IgM antiSRBC antibody than did XID mice given the control diet and approximately the same amount as the immunocompetent controls. However, the added dietary inositol did not have a significant effect on primary or secondary IgG responses to SRBC that remained impaired in the immunodeficient strain. These results suggested that myo-inositol in the doses given may modulate B-cell IgM responses by acting within the inositol second messenger system, either downstream from Bruton's tyrosine kinase or through another pathway(s) to induce an increased rate of proliferation and/or secretion by IgM PFC.

Reported below are the results of similar in vitro and in vivo studies in immunocompetent and XID mice of the effects on the primary responses to SRBC of three other agents known to interact within the inositol second messenger system: heparin (4), lithium chloride (5), and the inositol isomer, scyllo-inositol (6).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental Animals.
In these experiments, 12- to 20-week-old male and female B10.A(15R) and B10.A(18R) mice and 12-week-old CBA/CaHN-XID/J males (Jackson Laboratory, Bar Harbor, ME) were used. The mice were housed four to five per cage in an AAALAC-approved facility and given food and water ad libitum.

Diets.
The mice were fed standard mouse chow (Purina 5001) or the same diet supplemented with 0.4% (4 mg/g) myo-inositol (tissue culture grade; Sigma Chemical Co., St. Louis, MO) as previously described (1).

In Vitro Immune Response.
Spleens were removed aseptically from donor mice, and the cells were gently dissociated in Hanks balanced salt solution, washed once, and suspended in culture medium at a concentration of 10–20 x 106 viable cells/ml. Spleen cells from 8 to 12 donors were pooled for each separate experiment.

All cultures were done in triplicate, and replicate samples generally were within 5% of the mean. The culture medium was RPMI-1640 supplemented with L-glutamine, 10% heat-inactivated fetal calf serum, and penicillin and streptomycin (100 U and 100 µg/ml, respectively).

Primary responses to SRBC were elicited as described previously (7). Briefly, an underlay of washed SRBC (2%) in 1.5 ml of agar (0.25%) was placed in 35 x 10 mM tissue culture dishes (Falcon Plastics, Oxnard, CA), and 3 ml of spleen cell suspension were added after the agar had hardened. Myo-inositol, scyllo-inositol, heparin (preservative free), and LiCl (all from Sigma Chemical Co.) were filter sterilized and added to the cultures at the indicated concentrations. The cells were cultured at 37°C in 5% CO2 in air for 1–2 days. Medium and cells then were carefully removed, and 1 ml of guinea pig complement (1:7, absorbed with SRBC, Sigma) was added to each dish. After 1.5 hr at 37°C and 2 hr at room temperature, the areas of focal hemolysis (i.e., foci or colonies) were counted.

PFC.
Cells from the spleens of mice immunized with SRBC in vivo (0.2 ml 20% SRBC intravenous) and cells taken from culture dishes at the termination of the experiments were assayed for individual direct (IgM) PFC by methods described previously (7). Briefly, cells were added to 0.5% agar containing 2% SRBC and incubated 1.5 hr at 37°C. Guinea pig complement (1:7) previously absorbed with SRBC was added to the plates, which were then incubated at 37°C for 1 hr, followed by 2 hr at room temperature. Individual direct antibody-forming cells (PFC) were counted.

Statistical Analysis.
Means were compared using the paired t test, Student's t test, and the Mann-Whitney two-sample test (NCSS Statistical Program; NCSS, Kayville, UT). Comparisons were made between the responses (foci or PFC) of the mice receiving the test agent in the diet and those given the control diet, expressed in each separate experiment as a ratio (test agent/control). The means of these ratios (3–11 separate experiments) were calculated, and the degree of significance of the differences in the responses were determined.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spleen cells from naïve immunocompetent and XID mice were cultured with myo-inositol (22 mM), scyllo-inositol (2.2 mM), lithium chloride (1.0 or 1.5 mM), or heparin (5 U/ml: optimal dose 2–8 U/ml, data not shown), and the number of foci and PFC were determined on Day 1 and/or 2 (Table I)Go. It was found that myo-inositol, scyllo-inositol, and LiCl at both concentrations increased the number of foci but not the PFC per focus detected after 1-day cultures of spleen cells from immunocompetent and XID mice. Control cultures of spleen cells from competent mice produced approximately six times more foci/107 cells than did XID spleen cells on Day 1 and three times more on Day 2. No significant differences in the responses of B10.A(18R) and B10.A(15R) to these agents were noted.


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  		Table I.   Effects of Myo-Inositol, Scyllo-Inositol, LiCl, and Heparin on the Generation of Direct AntiSRBC Foci and PFC in 1- and 2-day Cultures of Spleen Cells from Naïve Immunocompetent and Immunodeficient (XID) Micea
 
The addition of heparin to the cultures resulted in an increase of PFC per focus but not total foci in cultures of spleen cells from XID mice on Day 1 and of foci and PFC per focus in cultures of cells from compentent mice on Day 2.

The above agents in all combinations were added to cultures of spleen cells from competent mice at the concentrations noted above, and the number of foci and PFC were determined on Days 1 and 2. In no instance did these combinations of agents significantly inhibit or enhance the responses to SRBC as compared with the control cultures (Table II)Go. Uniquely, in cultures containing heparin, alone or in combination with other agents, the donor spleen red blood cells were arranged on the agar in a "bullseye" pattern.


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  		Table II.   Effects of Myo-Inositol, Lithium Chloride, Heparin, and Mixtures of These Agents on the Generation of AntiSRBC Foci and PFC by Spleen Cells from Naïve Donors on the Second Day in Culturea
 
Immunocompetent mice injected with 50 U heparin at the time they were challenged with SRBC had significantly more PFC per spleen than did the controls on Days 3 and 4 but not 3 days later (Table III)Go. Heparin did not affect the indirect:direct PFC ratio in this study (unpublished data).


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  		Table III.   Effect of Heparin (50 USP units) Given ip with SRBC on the Number of Spleen PFC 3, 4, and 7 Days Later
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A previous report has shown in vivo and in vitro that pharmacological doses of myo-inositol appear to push naïve B cells from competent and immunodeficient XID mice toward terminal differentiation, accelerating and amplifying the IgM response but having little or no effect on the early conversion of IgG PFC (1). It was postulated that the effect of myo-inositol or a metabolite was expressed through modulation of the inositol second messenger system, a system also affected by heparin (8, 9) and lithium (10).

Myo-inositol 1,4,5-triphosphate (IP3) is a ubiquitous second messenger that couples agonist stimulation of a wide variety of cell-surface receptors to the mobilization of intracellular calcium (11, 12). IP3 is released from phosphatidylinositol in response to activation of certain receptor-associated or cytoplasmic tyrosine kinases. The generation of IP3 is critical to signal transduction through the B-cell antigen receptor (13, 14). Lithium interacts with the system by inhibiting inositol-1-phosphatase (12). Heparin can inhibit the response in cell-free or cell-membrane–permeable systems by blocking certain IP3 receptors (8, 9). Unlike myo-inositol and lithium that are actively transported across the plasma membrane (15), heparin normally does not enter cells. However, it has been shown that heparin can act extracellularly to modulate receptor activation by dimerization of certain ligands (16, 17).

In the studies reported here it was found that myo-inositol and scyllo-inositol at one-tenth the concentration were equally effective in increasing the number of specific PFC. Myo-inositol, scyllo-inositol, and lithium chloride accelerated the appearance of direct foci in cultures of spleen cells from normal and XID mice. When heparin was added to cultures of XID spleen cells, PFC were found to be increased on Day 1; however, PFC and foci were not increased in cultures of spleen cells from competent mice until 1 day later. This same temporal difference has been noted in vivo in XID and competent mice fed an inositol-supplemented diet and challenged with SRBC (unpublished data). The significance of these temporal differences, if real, is not clear although possibly it could reflect a modulating effect on the part of Bruton's tyrosine kinase that is deficient in XID mice (18).

The addition of combinations of these agents to cultures of spleen cells had no positive or negative effect on the generation of foci or PFC. These results were unexpected and are unexplained, although it is possible that at the doses used these agents modulate B-cell responses through secondary rather than primary signaling pathways where they are mutually inhibitory.

In summary, the results suggest that these agents, when acting alone, modulate B-cell responses by increasing the rate of proliferation and/or secretion through a signaling pathway(s) distal to, or more likely, independent of Bruton's tyrosine kinase. The evidence suggests that the mechanism is not the same with each agent.


   Footnotes
 
This work was supported in part by the Department of Veterans Affairs.

1 To whom requests for reprints should be addressed at VA Medical Center West Los Angeles, 11301 Wilshire Blvd., Los Angeles, CA 90073. E-mail: tyan.marvin_1+@west_la.va.gov Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tyan ML. Modulation of the antibody response to sheep red blood cells in normal and immunodeficient XID mice by myo-inositol. Proc Soc Exp Biol Med 215:258–263, 1997.[Abstract]
  2. Rawlings DJ, Saffran DC, Tsukada S, Largaespada DA, Grimaldi JC, Cohen L, Mohr RN, Bazan JF, Howard M, Copeland NG, Jenkins NA, Witte ON. Mutation of unique region of Bruton's tyrosine kinase in immunodeficient XID mice. Science 261:358–361, 1993.[Abstract/Free Full Text]
  3. Thomas JD, Sideras P, Smith CIE, Vorechovsky I, Chapman V, Paul WE. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261:355–358, 1993.[Abstract/Free Full Text]
  4. Short AD, Klein MG, Schneider MF, Gill DL. Inositol 1,4,5-trisphosphate-mediated quantal Ca2+ release measured by high-resolution imaging of Ca2+ within organelles. J Biol Chem 268:25887–25893, 1993.[Abstract/Free Full Text]
  5. Berridge MJ, Irvine RF. Inositol phosphates and cell signaling. Nature 341:197–205, 1989.[Medline]
  6. Sherman WR, Stewart MA, Kurien MM, Goodwin SL. The measurement of myo-inositol, myo-inosose-2, and scyllo-inositol in mammalian tissues. Biochem Biophys Acta 158:197–205, 1968.[Medline]
  7. Tyan ML, Ness DB, Gibbs PR. Fetal lymphoid tissues: Antibody production in vitro. J Immunol 110:1170–1173, 1973.
  8. Hill TD, Berggren PD, Boynton AL. Heparin inhibits inositol triphosphate–induced calcium release from permeabolized rat liver cells. Biochem Biophys Res Commun 149:897–901, 1987.[Medline]
  9. Gosh TK, Eis PS, Mullaney JM, Ebert CL, Gill DL. Competitive, reversible, and potent antagonism of inositol 1,4,5-triphosphate–activated calcium release by heparin. J Biol Chem 263:11075–11079, 1988.[Abstract/Free Full Text]
  10. Berridge MJ, Downes CP, Hanley MR. Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem J 206:587–595, 1982.[Medline]
  11. Berridge MJ. Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Annu Rev Biochem 56:159–193, 1987.[Medline]
  12. Berridge MJ. Inositol trisphosphate and calcium signaling. Nature 361:315–325, 1993.[Medline]
  13. Hata A, Sabe H, Kurosaki T, Takata M, Hanafusa H. Functional analysis of Csk in signal transduction through B-cell antigen receptor. Mol Cell Biol 14:7306–7313, 1994.[Abstract/Free Full Text]
  14. Takata M, Sabe H, Hata A, Iriaza T, Homma Y, Nukada T, Yamamura H, Kurosaki T. Tyrosine kinases Lyn and Syk regulate B-cell receptor–coupled Ca2+ mobilization through distinct pathways. EMBO J 13:1341–1349, 1994.[Medline]
  15. Holub BJ. Metabolism and function of myo-inositol and inositol phospholipids. Annu Rev Nutr 6:563–597, 1986.[Medline]
  16. DiGabriele AD, Lax I, Chen DI, Svahn CM, Jaye M, Schlessinger J, Hendrickson WA. Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature 393:812–817, 1998.[Medline]
  17. Weiss A, Schlessinger J. Switching signals on or off by receptor dimerization. Cell 94:277–280, 1998.[Medline]
  18. Kawakami Y, Hartman SE, Holland PM, Cooper JA, Kawakami T. Multiple signaling pathways for the activation of JNK in mast cells: Involvement of Bruton's tyrosine kinase, protein kinase C, and JNK kinases, SEK1 and MKK7. J Immunol 161:1795–1802, 1998.[Abstract/Free Full Text]
Received for publication August 16, 1999. Accepted for publication February 25, 2000.





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